B - CHAPTER V: ANALYTICAL METHODS & INSTRUMENTATION Flashcards
1
Q
– radiant energy; photons of energy travelling in a wavelike manner
A
ELECTROMAGNETIC ENERGY
2
Q
- The shorter the [?], the higher the [?]
A
wavelength; electromagnetic energy
3
Q
Types of Electromagnetic energies:
A
- Cosmic rays
- Gamma rays
- X-rays
- Visible
- Ultra-violet (UV)
- Infrared (IR)
- Radio, TV, microwave, etc.
4
Q
- distance between two peaks as the light travels in a wavelike manner
A
WAVELENGTH
5
Q
wavelength is expressed in
A
nanometers (nm), angstroms (Å), and millimicron (mµ)
6
Q
- 1 nm = [?] = [?]
A
10 Å
1 mµ
7
Q
Kinds of Wavelength:
A
- Visible spectra
- Non-visible spectra
8
Q
= 340 nm – 700 nm
A
Visible spectra
9
Q
(ultraviolet region)
A
= below 340 nm
10
Q
(infrared region)
A
= above 700 nm
11
Q
Kinds of Colorimetry:
A
- Visual Colorimetry
- Photoelectric Colorimetry
12
Q
– relies on visual acuity to determine end-point
A
Visual Colorimetry
13
Q
180 - 220
Short UV
A
14
Q
220 - 340
Short UV
A
15
Q
340 - 430
Visible
A
Violet
Yellow green
16
Q
430 - 475
Visible
A
Blue
Yellow
17
Q
475 - 495
Visible
A
Green blue
Orange
18
Q
495 - 505
Visible
A
Blue green
Red
19
Q
505 - 555
Visible
A
Green
Purple
20
Q
555 - 575
Visible
A
Yellow green
Violet
21
Q
575 - 600
Visible
A
Yellow
Blue
22
Q
600 - 620
Visible
A
Orange
Green blue
23
Q
620 - 700
Visible
A
Red
Blue green
24
Q
– measurement of light intensity in a much narrower wavelength
A
SPECTROPHOTOMETRY
25
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
SPECTROPHOTOMETRY
26
– measurements of light intensity of multiple wavelength
FILTER PHOTOMETRY
27
It makes use of filters (interference or transmission) to isolate part of the spectrum
FILTER PHOTOMETRY
28
Light passes through a [?] to provide a selection of the desired region of the spectrum to be used for measurements.
monochromator
29
are used to isolate a narrow beam of light and to improve its chromatic purity.
Slits
30
The light next passes through an [?] where a portion of the [?] is absorbed, depending upon the of the solution.
absorption cell
radiant energy
nature and concentration
31
Any light not absorbed is transmitted to a [?], which converts the light energy to [?] that can be registered on a [?] or a [?].
detector
electrical energy
meter/digital read-out
32
BEER’S LAW: The [?] is directly proportional to the [?] and inversely proportional to the [?]
concentration of the solution
amount of light absorbed
logarithm of transmitted light
33
%T = [?]
Absorbance / Optical Density (O.D.) =
ratio of the radiant energy transmitted, divided by the radiant energy incident on the sample
the amount of light absorbed
34
BOUGUER’S LAW or LAMBERT’S LAW: [?] is directly proportional to the [?] of light path
Absorbance
length
35
? = absorbance
A
36
? = 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
37
[?] = length of light path in cm
b
38
[?] = molar concentration of absorbing substance
c
39
Internal Parts of the Spectrophotometer
source
entrance slit
despersion device
exit slit
sample
detector
40
A – Light Source
E – Cuvet
B – Entrance slit
F – Detector
C – Monochromator
G – Meter
41
– provides a continuous spectrum of white light which can be separated at different wavelengths
LIGHT SOURCE
42
– isolates a narrow beam of radiant energy; prevents stray light from entering the monochromator
ENTRANCE SLIT
43
– wavelength selector; isolates radiant energy of desired wavelength and excluding that of other wavelengths
MONOCHROMATOR
44
– used to hold the solution whose concentration is to be measures
ANALYTICAL / ABSORPTION CELL / CUVETTE
45
– measure light intensity by converting light signal into electrical signal
DETECTORS
46
– electrical energy from a detector is displayed
READ-OUT DEVICES
47
TYPES OF LIGHT SOURCE
A. Tungsten Iodide lamp
B. Quartz Halide lamp
C. Deuterium Discharge lamp
D. Infrared Energy source
E. Mercury Vapor lamp
F. Hollow Cathode lamp
48
TYPES OF MONOCHROMATOR
A. Prism
B. Diffraction Gratings
C. Transmission Filters
D. Interference Filters
49
TYPES OF ANALYTICAL / ABSORPTION CELL / CUVETTE
a. Borosilicate glass
b. Quartz or plastic
c. Alumina silica glass
50
TYPES OF DETECTORS
a. Barrier-Layer cell (Photocell or Photovoltaic cell)
b. Photoemissive tube or Phototube
c. Photoconductive tube or Photoresistive tube
d. Photomultiplier tube
51
– produces energy wavelength from 340 – 700 nm (visible region); used for moderately diluted solution
A. Tungsten Iodide lamp
52
– contains small amounts of halogen such as iodine to prevent the decomposition of the vaporized tungsten from the very hot filament
B. Quartz Halide lamp
53
– provides energy source with high output in the UV range (down to 165 nm)
C. Deuterium Discharge lamp
54
– used above 800 nm
D. Infrared Energy source
55
– emits narrow bands of energy at well-defined places in the spectrum (UV and visible)
E. Mercury Vapor lamp
56
– consists of a gas-tight chamber containing anode, a cylindrical cathode, and inert gas such as helium or argon
F. Hollow Cathode lamp
57
Infrared Energy source Examples:
Merst glower
Globar
58
– an electrically heated rod of rare earth element oxides
Merst glower
59
– uses silicon carbide
Globar
60
– wedge-shaped pieces of glass, quartz, NaCl, or some other material that allows transmission of light
Prism
61
o Disperses white light into a continuous spectrum of colors by refraction
Prism
62
o Produces a non-linear spectrum.
Prism
63
are for visible region while quartz prisms are for the UV region
o Glass prisms
64
The (?) are close to each other and those of (?) are widely spaced.
longer wavelengths
shorter wavelengths
65
– 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
Diffraction Gratings
66
o Rays of radiant energy bend (refract) around the sharp edges of the grooves
Diffraction Gratings
67
o Extent of refraction varies with the wavelength
Diffraction Gratings
68
– colored glass or colored gelatin sandwiched between two glass plates
Transmission Filters
69
o Light outside the transmission band are absorbed by the colored material
Transmission Filters
70
o Band pass is 35 – 50 nm or more
Transmission Filters
71
– dielectric material (e.g. NaF) sandwiched between two half-silvered pieces of glass
Interference Filters
72
o The thickness of the layer determines the wavelength of energy transmitted.
Interference Filters
73
o Band pass is 10 – 20 nm
Interference Filters
74
– for solutions that do not etch glass
a. Borosilicate glass
75
– does not absorb UV radiation at wavelength below 320 nm
b. Quartz or plastic
76
– good for 340 nm and above (visible region)
c. Alumina silica glass
77
- Composed of a film of light sensitive material (e.g. Selenium) on an iron plate with a transparent layer of silver
a. Barrier-Layer cell (Photocell or Photovoltaic cell)
78
- 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. Barrier-Layer cell (Photocell or Photovoltaic cell)
79
- Has photosensitive material that gives off electrons when light energy strikes it
Photoemissive tube or Phototube
80
- Consists of 2 electrodes (cathode and anode) sealed in an evacuated glass
Photomultiplier tube
81
- A device whose electrical resistance decreases as the level of incident light is raised
Photoconductive tube or Photoresistive tube
82
- Cadmium sulfide or cadmium selenide are the light-sensitive materials typically used for the visible region
Photoconductive tube or Photoresistive tube
83
- Does not require an external power source
Photoconductive tube or Photoresistive tube
84
- Capable of significantly amplifying a current
Photomultiplier tube
85
- The cathode is a negative light-sensitive metal that absorbs light and emits electrons in proportion to the radiant energy that strikes the surface
Photomultiplier tube
86
- Electrons go to the dynodes, where electrons produce 4 – 6 additional electrons
Photomultiplier tube
87
- The electrons are collected at a final electrode, the positive anode
Photomultiplier tube
88
Photomultiplier tube Advantages:
- rapid response time
- very sensitive
- low fatigue
89
– electrical energy from a detector is displayed
READ-OUT DEVICES
90
– the output of the detector is used to drive a sensitive meter directly without further amplification
a. Direct reading system
91
– the output of the detector is balanced against the output of a reference circuit
b. Null Point System
92
– numerical display of absorbance or converted values of concentrations
c. Digital Read-out
93
K
94
DOUBLE BEAM SPECTROPHOTOMETERS
1. Double Beam-In-Space
2. Double Beam-In-Time
95
• All components are duplicated except the light source
Double Beam-In-Space
96
• The beams of light pass through different components but at the same time
Double Beam-In-Space
97
• Uses a light beam chopper (a rotating wheel) – with alternate silvered sections and cut out sections, inserted after the exit slit
Double Beam-In-Time
98
It involves the measurement of emitted light when electrons in an atom become excited by heat energy produced by the flame.
FLAME PHOTOMETRY / FLAME EMISSION SPECTROPHOTOMETRY / FILTER PHOTOMETRY
99
When these electrons return to their ground state, they emit light characteristic of the ions present.
FLAME PHOTOMETRY / FLAME EMISSION SPECTROPHOTOMETRY / FILTER PHOTOMETRY
100
It is used primarily to determine concentration of sodium, potassium or lithium since these alkali metals are easy to excite
FLAME PHOTOMETRY / FLAME EMISSION SPECTROPHOTOMETRY / FILTER PHOTOMETRY
101
Sodium =
yellow
102
Rubidium =
red
103
Potassium =
violet
104
Magnesium =
blue
105
Lithium =
red
106
COMPONENTS of the FLAME PHOTOMETER
1. GASES
2. BURNER ASSEMBLY
3. INTERFERENCE FILTERS as MONOCHROMATOR
4. DETECTOR
107
Types of Burner:
A. Total Consumption Burner
B. Premix Burner
108
mixture of hydrogen and oxygen gas
- acetylene
- propane
- natural gas
109
BURNER ASSEMBLY
a. Aspirator
b. Atomizer (Nebulizer )
c. Flame
110
– draws sample into the flame
a. Aspirator
111
– creates a fine spray of sample solution to be fed into the flame of the burner
b. Atomizer (Nebulizer )
112
– provides heat energy for excitation
c. Flame
113
– aspirate sample directly into the flame, the gases are passed at high velocity over the end of the capillary suspended in the solution
A. Total Consumption Burner
114
– 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
B. Premix Burner
115
- transmit yellow light (589 nm)
Na filter
116
- transmit violet light (767 nm)
K filter
117
- transmit red light (761 nm)
Lithium
118
DETECTOR – uses photocell as detector
FLAME PHOTOMETRY
119
The Internal Standard in Flame Photometry: Uses (?)
Lithium or Cesium
120
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 .
chemical bond
lower energy level
light source; element
121
- Measures the amount of light absorbed by ground state atom
ATOMIC ABSORPTION SPECTROPHOTOMETRY (AAS)
122
COMPONENTS OF ATOMIC ABSORPTION SPECTROPHOTOMETRY (AAS)
1. LIGHT SOURCE
2. MECHANICAL ROTATING CHOPPER
3. BURNER
4. MONOCHROMATOR
5. DETECTOR
6. METER or READ-OUT DEVICE
123
– hollow cathode lamp, which produces a wavelength of light specific for the kind of metal in the cathode
1. LIGHT SOURCE
124
– modulates light beam coming from the hollow cathode lamp
2. MECHANICAL ROTATING CHOPPER
125
– uses flame to dissociate the chemical bonds and form free, unexcited atoms
3. BURNER
126
Two types of Burner:
a. Total Consumption burner
b. Pre-mix burner
127
– flame is more concentrated and can be made hotter, thus lessening chemical interferences.
a. Total Consumption burner
128
– gases are mixed and the sample is atomized before entering the flame and the large droplets go to waste and not in the flame.
b. Pre-mix burner
129
It has less noisy signals with longer pathlength and greater absorption and sensitivity.
b. Pre-mix burner
130
– selects the desired wavelength from a spectrum of wavelength which could either be a prism or a diffraction grating.
4. MONOCHROMATOR
131
– uses photomultiplier tubes to measure the intensity of the light signal.
DETECTOR
132
– 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.
FLUORESCENCE
133
COMPONENTS OF FLUORESCENCE SPECTROPHOTOMETRY
1. LIGHT SOURCE
2. MONOCHROMATORS:
a. Primary filter / Excitation filter
b. Secondary filter / Emission filter
3. PHOTOMULTIPLIER
4. READ-OUT DEVICE
134
– hydrogen discharge lamp or xenon lamp
1. LIGHT SOURCE
135
2. MONOCHROMATORS:
a. Primary filter / Excitation filter
b. Secondary filter / Emission filter
136
– isolates the ultraviolet light
a. Primary filter / Excitation filter
137
– isolates secondary emission (filter, prism or diffraction grating)
b. Secondary filter / Emission filter
138
– separation of a substance in a pure form and then determining its dry weight
GRAVIMETRIC METHOD
139
GRAVIMETRIC METHOD Example:
Total Lipid determination
140
– the unknown sample is made to react with a known solution (titrating agent) in the presence of an indicator
VOLUMETRIC / TITRIMETRIC METHOD
141
VOLUMETRIC / TITRIMETRIC METHOD Example:
Chloride determination (Schales & Schales)
142
– measurement of the amount of light blocked by a particulate matter suspended in solution (180° to the incident beam)
TURBIDIMETRY
143
Factors affecting turbidimetry:
o Size and number of particles
o The depth of the tube
o Cross-sectional area of each particle
144
– 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)
NEPHELOMETRY
145
– 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)
NEPHELOMETRY
146
• The factors affecting turbidimetric measurements are the same factors affecting (?) measurements
nephelometric
147
• It is more specific than turbidimetry
NEPHELOMETRY
148
– it is used to measure the disintegration of a radioisotope per minute
SCINTILLATION COUNTER
149
Types of Radiation:
1. Alpha
2. Beta
3. Gamma
150
– positively charged particles; resemble the nucleus of helium atom with a mass of 4 o Have very little energy
1. alpha
151
– resembles an electron with both negative (β-) and positive (β+) charges but essentially no mass o Exists in two forms: soft and hard beta
2. Beta
152
– a form of electromagnetic energy with no mass, only energy o Exists in two forms: soft and hard gamma
3. Gamma
153
Types of Scintillation Counters:
1. Solid Scintillation Counter
2. Liquid Scintillation Counter
154
– measures gamma radiation using thallium activated NaI crystal as scintillator and PM tube as detector with preamplifier circuit
1. Solid Scintillation Counter
155
– measures beta radiation using liquid flour as scintillator
2. Liquid Scintillation Counter
156
– an immunologic procedure involving the use of radioisotope
RADIOIMMUNOASSAY
157
Substances involved in RIA:
1. Unlabelled antigen (Ag)
2. Radiolabelled antigen (Ag)
3. Antibody
158
– substance being analyzed
1. Unlabelled antigen (Ag)
159
– acts as label 3.
2. Radiolabelled antigen (Ag)
160
– provide binding site for the two antigens
3. Antibody
161
Types of RIA:
1. Solid RIA
2. Liquid RIA
162
– measurement of differences in voltage at a constant current
POTENTIOMETRY
163
o The unknown voltage introduced into the potentiometer circuit opposes a known reference voltage
POTENTIOMETRY
164
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
POTENTIOMETRY
165
o The relationship between the measured voltage and the sought-for concentration
POTENTIOMETRY
166
The relationship between the measured voltage and the sought-for concentration is shown by the Nernst Equation
POTENTIOMETRY
167
– measurement of differences in current at a constant voltage
POLAROGRAPHY
168
o Used to measure trace metals, oxygen, Vit. C, and amino acid concentration
POLAROGRAPHY
169
o The relationship between the differences in current and voltage is shown by the Ilkovic Equation
POLAROGRAPHY
170
– the measurement of the amount of electricity (in coulombs) at a fixed potential
COULOMETRY
171
is equal to a current flow of 1 ampere per second
coulomb
172
o The (?) consumed can be related directly to the concentration of the unknown
number of coulombs
173
o The relationship is expressed by the Faraday’s Law
COULOMETRY
174
– measurement of the amount of current that flows when a constant voltage is applied to the measuring electrode
AMPEROMETRY
175
– measurement of the current flow between two non-polarizable electrodes between which a known electrical potential is established
CONDUCTOMETRY
176
• Separation is based on solubility
PRECIPITATION
177
The precipitate is studied by:
a. Turbidimetric method
b. Chemical reaction (after being dissolved)
c. Gravimetric method
178
- separates dissolved materials
ULTRAFILTRATION
179
- removes particulate matter
ULTRAFILTRATION
180
- separation of dissolved molecules
DIALYSIS
181
- movement through a semi-permeable membrane driven by a force or pulled through a vacuum
ULTRAFILTRATION
182
- movement through a semi-permeable membrane more freely
DIALYSIS
183
Cellulose esters
Cellulose acetate Polyamide
Polyvinyl chloride
Sheets, disks or hair thin fibers, conical
ULTRAFILTRATION
184
Cellophane (regenerated cellulose) in sheets or tubing
DIALYSIS
185
- For desalting, fractionation of protein solutions and the preparation of pff
ULTRAFILTRATION
186
- Employed in continuous flow automated systems
DIALYSIS
187
- used to purify or concentrate samples
DIALYSIS
188
CHROMATOGRAPHY Requires two (2) phases:
(1) Solid support
(2) Mobile phase
189
– coated or uncoated
(1) Solid support
190
– flowing gas or liquid
(2) Mobile phase
191
TWO GENERAL TYPES of CHROMATOGRAPHY:
A. Adsorption Chromatography
B. Partition Chromatography
192
– molecules separated are adsorbed at the surface of a solid support or flow with the mobile phase
A. Adsorption Chromatography
193
– solid support is coated with a film of water or non-volatile organic liquid
B. Partition Chromatography
194
Partition Chromatography Examples:
TLC, GLC
195
1. Paper Chromatography
a. Solid or immobile phase
b. Mobile phase
196
– paper is composed of cellulose; the matrix of cellulose is bound to water
a. Solid or immobile phase
197
– organic solvent
b. Mobile phase
198
o involves partition between water and organic solvent
Mobile phase
199
o if the molecules are more soluble in the flowing solvent, the faster it will move along the paper
Mobile phase
200
o if the molecules are more soluble in water, they do not move very fast
Mobile phase
201
Rf = ratio of the distance of movement by a (?) to the distance of the (?) front
compound
solvent
202
Rf = a/B
Where:
a = distance travelled by (?) from origin to front of spot
b = distance travelled by (?)
compound
solvent
203
o Uses a flat sheet of chromatographic material
Thin Layer Chromatography
204
Thin Layer Chromatography Advantages over paper chromatography
• Solid Support
• Mobile Phase
205
Thin Layer Chromatography
• Solid Support: water bound to
a. Silica or silicic acid
b. Alumina – aluminum oxide + aluminum hydroxide
206
Thin Layer Chromatography
• Mobile Phase:
organic solvent
207
Thin Layer Chromatography
Qualitative analysis:
based on colors and positions
208
Thin Layer Chromatography
Quantitative analysis:
remove spots and extract
209
o Separation is based on electrical charge
Ion-Exchange Chromatography
210
– capture anions
o Anion exchangers
211
– capture cations
o Cation exchangers
212
Ion-Exchange Chromatography
• Stationary / Immobile / Solid support
a. Aluminum silicate
b. Polysaccharide
c. Synthetic resins – polystyrene beads
213
Ion-Exchange Chromatography
• Mobile Phase:
water
214
o Separation is based on differences in molecular size
Gel Filtration / Molecular Sieve / Gel Permeation / Size Exclusion / Molecular Exclusion
215
Gel Filtration / Molecular Sieve / Gel Permeation / Size Exclusion / Molecular Exclusion
• Stationary phase
a. Polyacrylamide (plastic)
b. Sephadex (cross-linked polysaccharide)
c. Porous beads
216
Gel Filtration / Molecular Sieve / Gel Permeation / Size Exclusion / Molecular Exclusion
• Mobile phase:
flowing water
217
Gas Chromatography
Two general types:
a. Gas – Liquid Chromatography (GLC)
b. Gas – Solid Chromatography (GSL)
218
Gas Chromatography
• Stationary Phase –
diatomaceous earth (silica) coated with a non-volatile organic liquid = silicone polymer or alcoholic wax
219
Gas Chromatography
– inert carrier gas (Helium or Nitrogen)
• Mobile Phase
220
a. Gas – Liquid Chromatography (GLC) –
b. Gas – Solid Chromatography (GSL) –
based on partition
based on adsorption
221
❖ Involves the migration of charged solutes or particles in a supporting medium under the influence of an electric field
ELECTROPHORESIS
222
– migration of small ions or molecules
• Iontophoresis
223
– migration of charged macromolecules in a porous medium such as cellulose acetate, paper or agarose
• Zone electrophoresis
224
➢ it generates an ELECTROPHORETOGRAM – a display of protein zones
• Zone electrophoresis
225
– a display of protein zones
ELECTROPHORETOGRAM
226
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 (?)
zwitterion
CATHODE
ANODE
227
Migration depends on:
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
STAINS:
Protein Stains
Isoenzymes: Nitrotetrazoleum Blue
Lipoprotein
229
Protein Stains
o Amido Black
o Bromphenol Blue
o Coomasie Brilliant Blue
o Nigrosin
o Ponceau S
230
Lipoprotein
o Fat Red 7B (Sudan Red)
o Oil Red O
o Sudan Black B
231
TYPES of ELECTROPHORESIS:
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
ISOELECTRIC FOCUSING An electrophoretic method in w/c proteins are separated on the basis of their pI (isoelectric pH)
233
An electrophoretic method in w/c proteins are separated on the basis of their pI (isoelectric pH)
ISOELECTRIC FOCUSING
234
Makes use of the property of proteins that their net charges are determined by the pH of their local environment
ISOELECTRIC FOCUSING
235
Proteins show considerable variation in pI, but pI values fall in the range pH 3–12 (many having pIs between pH 4–7)
ISOELECTRIC FOCUSING
236
1. Establishing pH gradients
Accomplished w/ the use of:
a. Carrier Ampholytes (Amphoteric electrolytes)
b. Acrylamide buffers
2. Gel for Isoelectric Focusing
237
- Mixtures of molecules containing multiple aliphatic amino & carboxylate groups (buffer molecules)
Carrier Ampholytes (Amphoteric electrolytes)
238
- Included directly in IEF gels
Carrier Ampholytes (Amphoteric electrolytes)
239
- Derivatives of Acrylamide containing both reactive double bonds & buffering groups
Acrylamide buffers
240
- Covalently incorporated in PAG at the time of casting
Acrylamide buffers
241
Gel for Isoelectric Focusing
❖ Polyacrylamide Gel
242
– large-pore convective matrices - Polymerized with an initiator system including Riboflavin for photo-polymerization
❖ Polyacrylamide Gel
243
– introduction of the 1st automated analyzer by Technicon
- Continuous-flow
1957
244
: sequential batch analyzer capable of providing single test result on approx. 40 samples / hour
- Continuous-flow
245
– Technicon instruments w/c were next developed
Simultaneous Multiple Analyzer (SMA)
246
- With multiple channels (for diff. tests)
Simultaneous Multiple Analyzer (SMA)
247
- 6-12 test results simultaneously at the rate of 360
Simultaneous Multiple Analyzer (SMA)
248
– 720 tests per hour
Simultaneous Multiple Analyzer (SMA)
249
– specialty area w/ rapidly developing arsenal of analyzers
IMMUNOCHEMISTRY
250
- Immunological techniques for assaying drugs, specific proteins, tumor markers & hormones
IMMUNOCHEMISTRY
251
- Fluorescence Polarization Immunoassay
- Nephelometry
- Chemiluminescent Detection
IMMUNOCHEMISTRY
252
BASIC APPROACHES OF AUTOMATED ANALYZERS
I. CONTINUOUS - FLOW
II. CENTRIFUGAL ANALYSIS
III. DISCRETE ANALYSIS
253
- Liquids (reagents, diluents & samples) are pumped through a system of continuous tubing
CONTINUOUS - FLOW
254
- Samples are introduced in a sequential manner, following each other through the same network
CONTINUOUS - FLOW
255
- Batch analysis can be used (e.g. large # of specimen in one run)
CONTINUOUS - FLOW
256
More sophisticated continuous flow anayzers Use parallel single channels to run multiple tests on each sample (e.g. SMA & SMAC)
CONTINUOUS - FLOW
257
Major drawbacks: significant carry-over problems & wasteful use of continuously flowing reagents
CONTINUOUS - FLOW
258
- Uses the force generated by centrifugation to transfer & then contain liquids in separate cuvets for measurement
CENTRIFUGAL ANALYSIS
259
- Capable of running multiple samples, one test at a time, in a batch MAJOR ADV.: batch analysis (e.g. COBAS - Bio by Roche Diagnostics)
CENTRIFUGAL ANALYSIS
260
– Bio by Roche Diagnostics)
CENTRIFUGAL ANALYSIS
261
- Most popular & versatile
DISCRETE ANALYSIS
262
- Separation of each sample & accompanying reagents in a separate container
DISCRETE ANALYSIS
263
- Capability of running multiple tests one sample at a time OR multiple samples one test at a time
DISCRETE ANALYSIS
264
- Random access, stat capabilities
DISCRETE ANALYSIS
265
Use parallel single channels to run multiple
tests on each sample (e.g. SMA & SMAC)
CONTINUOUS - FLOW
