PRELIM LEC: ANALYTICAL TECHNIQUES AND INSTRUMENTATION (TRANSES) Flashcards
TRANSES AND NOTES
1
Q
-are described as photons of
energy traveling in waves
A
Electromagnetic Radiation
2
Q
is the highest point while trough is the lowest point
A
Crest or peak
3
Q
is the distance of two peaks/crest or troughs
when light travels in a wavelike manner
A
Wavelength
4
Q
Units:
A
Angstrom (A)Millimicrons (mu) Nanometer (nm)
5
Q
Conversion factors:
A
1 nm = 10 A; 1 nm = 1 mu; 1 A = 10 mu
6
Q
is the number of waves that pass an observation point in a unit of time
A
Frequency
7
Q
is the distance between two adjacent peak and
trough
A
Amplitude
8
Q
is inversely proportional to the frequency of the light wave
A
Wavelength
9
Q
is inversely proportional to the wavelength of light
A
Energy
10
Q
The shorter the wavelength,
A
the larger number of photons will
be contained in a given distance
11
Q
More photons represents more energy, hence,
A
shorter wavelengths represent higher energy
12
Q
Wavelength or frequency of the electromagnetic waves are
perceived as
A
color or hue
13
Q
shorter wavelength = high frequency color
A
bluish colors
14
Q
long wavelength = low frequency colors
A
reddish color
15
Q
Height or amplitude of the electromagnetic waves are
perceived as
A
intensity or brightness
16
Q
great amplitude=
A
bright colors
17
Q
small amplitude =
A
dull colors
18
Q
two kinds of wavelengths:
A
Visible Spectra
Invisible Spectra
19
Q
can be observed at 340-700nm
A
Visible Spectra
20
Q
wavelength of less
than 400nm
Invisible Spectra
A
ultraviolet
21
Q
wavelength of greater than
700 nm
Invisible Spectra
A
Infrared
22
Q
Other types of electromagnetic energy includes:
A
Cosmic Rays, Gamma Rays, X-rays and Appliances (Radio, TV, Microwave, Etc.)
23
Q
Red (650-700)
Complementary Color
A
Green
24
Q
Orange (600-650)
Complementary Color
A
Blue
25
Yellow (550-600)
| Complementary Color
Indigo
26
Green (500-550)
| Complementary Color
Red
27
Blue (450-500)
| Complementary Color
Orange
28
Indigo (400-450)
| Complementary Color
Yellow
29
Violet (350-400)
| Complementary Color
Yellow
30
Principle: Measures the amount of light transmitted to determine the concentration of the light-absorbing substance in the solution; the measurement of the light transmitted by a solution to determine the concentration of the light-absorbing substance in the solution
Spectrophotometry
31
states that the concentration of substance is directly proportional to the amount of light absorbed but inversely proportional to the logarithm of transmitted light
Beer-Lamberts Law
32
ratio of the radiant energy transmitted, divided by the radiant energy incident on the sample
%T
33
amount of light
absorbed
Absorbance (OD-optical density)
34
provides electromagnetic radiation as visible, infrared, or UV light
Light source
35
TYPE OF LIGHT SOURCE:
1.Tungsten/Tungsten-iodide lamp
2.Mercury Vapor Lamp
3.Deuterium Discharge Lamp
4.Infrared Energy Source
5.Quart Halide Lamp
6.Hollow Cathode Lamp
36
ideal for emission of light within the visible region (iodide prolongs stability of Tungsten); produces energy wavelength from 340-700nm (visible region); used for moderately diluted solution
Tungsten/Tungsten-iodide lamp
37
* ]can emit UV ligh
* exists narrow bands of energy at well defined places in the spectrum UV and visible light
Mercury Vapor Lamp
38
energy wavelength UV
range (down to 165nm)
Deuterium Discharge Lamp
39
above 800 nm
Infrared Energy Source
40
contains small amt of halogen such as iodine to prevent the decomposition of vaporized tungsten
Quart Halide Lamp
41
consists of a gas-tight chamber containing anode, a cylindrical cathode and insert gas such as helium
Hollow Cathode Lamp
42
reduces stray light and prevents scattered
light from entering the monochromator
Entrance Slit
43
solates the specific wavelength of choice
Monochromator
44
TYPES OF MONOCHROMATOR:
1. Prism
2. Gratings
3. Colored Filters
4. Interference Filters
45
wedge-shaped pieces of glass, quartz, or sodium chloride that allows transmission of light wherein each side of the prism has different thickness allowing selection of wavelength of light
Prism
46
disperses white light into a continuous spectrum of colors based on variation of refractive index for different wavelength
Prism
47
has small grooves cut at such an angle that each grooves behave like a very small prism and the wavelengths are bent as they pass a sharp corner
Gratings
48
separate white light into various color comp
Gratings
49
made of glass that absorbs some portion of the electromagentic spectrum and transmit others wherein light energy is absorbed by dye components on the class and is dissipated as heat
Colored Filters
50
band pass is 35-50nm or more
Colored Filters
51
enhances desired wavelength by constructive
interference and eliminates others by destructive interferences
Interference Filters
52
utlizes the wave cx of light to enhance the intensity of the desired wavelength by constructive interference and eliminates others by destructive interference and reflections
Interference Filters
53
band pass is 10-20nm
Interference Filters
54
where the light from the monochromator will pass through
exit slit
55
used to hold the solution in the instrument
whose concentration is to be measured
Analytical Cell
56
TYPES OF ANALYTICAL CELLS:
1. Borosilicate Glass
2. Quartz or Plastic
3. Aluminum Silica Glass
4. Soft glass
57
alkaline solution that do not etch
glass
Borosilicate Glass
58
best for wavelength below 320nm
PLASTIC CELLS - BOTH VISIBLE AND UV RANGE
Quartz or Plastic
59
best for visible light
Aluminum Silica Glass
60
best for acidic solution
Soft glass
61
- converts transmitted light energy into an equivalent amount of electrical energ
Detector
62
TYPES OF DETECTOR:
1. Barrier layer cells
2. Photoemission tube
3. Photomultiplier tube
63
composed of film of light sensitive
material; no power source needed
Barrier layer cells
64
has photosensitive material that gives off electron when light energy strikes it; requires
an outside voltage for operation
Photoemission tube
65
used a series of electrodes to internally amplify the photosignal before leaving the tube
Photomultiplier tube
66
simplest method of displaying output of the detection system
Meter (read out device)
67
Principle: measures the light emitted when electrons in an atom become excited by heat energy produced by the flame
Flame Emission Spectrophotometry
68
Measures electrolytes with a 1+ charge:
Na, K, Li
69
Excited atoms return to ground state by emitting light energy that is characteristic of that atom
Flame Emission Spectrophotometry
70
Sodium filter- transmits only
yellow light (589 nm)
71
Potassium filter- transmits only
violet light (367 nm)
72
73
74
Lithium filter- transmits only
red light (767nm)
75
-breaks the chemical bonds to produce atoms
-source of energy that will be absorbed by the atoms to enter the excitation state
Flame
76
using a mixture of hydrogen and oxygen gas (acetylene, propane or natural gas)
Gases
77
breaks up the solution into finer droplets so that the atom will absorb heat energy from the flame and get excited
Atomizer or Burner
78
aspirate sample directly into
flame
Total Consumption Burner
79
80
involves the gravitational feeding of solution
Premix Burner
81
involves the gravitational feeding of solution
| FLAME EMISSION SPECTOPHOTOMETRY
Interference Filter
82
serves as photodetector
| FLAME EMISSION SPECTOPHOTOMETRY
Photocell
83
is the preferred internal standard which also acts as a radiation buffer
lithium
84
Criteria for Internal Standard
a. Concentration should be precisely the same in all samples and standards
b. Energy must be close to the required amount to excite the measured element
c. Normally found in solution being analyzed
85
Principle: measures concentration of the element by detecting absorption of electromagnetic radiation by atoms
Atomic Absorption Spectrophotometry
86
The elements are not excited but are dissociated from their chemical bonds and placed in the unionized, unexcited ground state
Atomic Absorption Spectrophotometry
87
Measures electrolytes with a 2+ charge:
| Atomic Absorption Spectrophotometry
Ca2+, Mg2+
88
hollow cathode lamp, which produces a
wavelength of light that is specific for the kind of metal in the cathode
| Components of AAS
Light Source
89
modulates light beam
coming from the light source
| Components of AAS
Mechanical Rotating Chopper
90
- uses flame to dissociate the chemical bonds and
form free unexcited atoms
| Components of AAS
Burner
91
ADV: flame is more concentrated and hotter
DISADV: produces large droplets in the flame; noisier
| Components of AAS
Total Consumption Burner
92
gases are mixed; sample is atomized before entering the flame
| Components of AAS
Premix Burner
93
ADV: greater absorption and sensitivity; less noisy; large droplets go to the waste
DISADV: flame is less ho
| Components of AAS
Premix Burner
94
selects the desired wavelength from a spectrum of wavelength
| Components of AAS
Monochromator
95
Interferences:
situation at which the flame could not
dissociate the sample into neutral atoms
| Components of AAS
Chemical
96
Interferences:
situation at which atoms in the flame
become excited and emits energy
| Components of AAS
Ionization
97
Principle: Unknown samples are made to react with a known solution in the presence of an indicator
Volumetry/Titrimetry
98
Volumetry/Titrimetry Sample tests:
Schales & Schales method;EDTA Titration
99
Principle: it is the isolation of the pure form of the sample and its derivatives and the determination of its dry weight
Gravimetry
100
Gravimetry Sample tests:
Lipid determination
101
Principle: Measures the amount of light blocked by a suspension of particular matter as light passes through the cuvette
Turbidimetry
102
Turbidimetry Factors affecting measurement:
o Size and number of particles
o Depth of the tube
o Cross-sectional area of each particle
103
Principle: Measures the amount of light scattered by small particles at an angle to the beam incident on the cuvette
Nephelometry
104
Factors affecting measurement same as turbidimetry
Nephelometry
105
Principle: measures multiple properties of cells suspended in a moving fluid medium
Flow Cytometry
106
Flow Cytometry Process:
a. All cells pass a single-file through a sensing point, where they are intercepted by a laser beam
b. Cell suspensions are introduced into the flow chamber.
c. As the cells will pass through the flow chamber, they are surrounded by low-pressure sheath that creates laminar flow forcing the specimen into the center.
d. Each of the cells is intersected by light
e. The laser light excites the dye which emits a color of light that is detected by the photomultiplier tube, or light detector.
f. The cells are then sorted based from their electrical charge
g. As the drop forms, an electrical charge is applied to the stream to form a charge
h. This charged drop is then deflected left or right by charged electrodes and into waiting sample tubes.
i. Drops that contain no cells are sent into the waste tube. The end result is three tubes with pure subpopulations of cells.
j. The number of cells is each tube is known and the level of fluorescence is also recorded for each cell.
107
INTERPRETATION:
cell size
| Flow Cytometry
Forward light scatter
108
INTERPRETATION:
cell granularity and nuclear irregularity
| Flow Cytometry
90° angle scatter
109
Principle: involves the separation of a mixture on the basis of specific differences of the physical and chemical characteristics of the different components on a supporting
medium
Chromatography
110
the constituents of the mixture are separated by a continuous redistribution between two phases:
| Chromatography
mobile phase
stationary phase
111
mobile phcarries the complex mixture
| Chromatography
mobile phase
112
where the mobile phase flows
| Chromatography
stationary phase
113
Types of Chromatorgraphy:
1. Paper Chromatography
2. Thin Layer Chromatography
3. Liquid-Liquid Chromatography
4. Ion Exchange Chromatography
5. Column Chromatography
6. Gel Chromatography
7. Gas Chromatography
114
Principle: A spot of the substance fractioned is placed on the paper just above the solvent level
Paper Chromatography
115
Basis of Separation:
| Paper Chromatography
o Rate of diffusion
o Solubility of solute
o Nature of the solvent
116
Same principle as paper chromatography but differs in
the sorbent used
Thin Layer Chromatography
117
thin plastic plates impregnated to a layer of
silica gel, alumina, polyacrylamide gel or starch gel
| Thin Layer Chromatography
SORBENT
118
Principle: separation of substances according to their solubility in an organic/non-polar solvent and in an aqueous/polar solvent
Liquid-Liquid Chromatography
119
“Like Dissolves Like”
| Liquid-Liquid Chromatography
o highly polar substance = more soluble in a
highly polar solvent (water)
o lesser polar substance = more soluble in a
less polar solvent (organic substance)
120
Clinical use:
| Liquid-Liquid Chromatography
fractionation of barbituates and lipids
121
Principle: the use of a resin (the stationary solid phase) is used to covalently attach anions or cations onto it
Ion Exchange Chromatography
122
Principle: adsorption of the solutes of a solution through a stationary phase and separates the mixture into individual components
Column Chromatography
123
Basis of Separation:
| Column Chromatography
o difference in pH
o polarity of solven
124
Clinical use:
| Column Chromatography
Fractionation of sugars
125
Principle: the use of a resin (the stationary solid phase) is used to covalently attach anions or cations onto it
Gel Chromatography
126
Basis of Separation:
| Gel Chromatography
o Molecular weight & size
o Charge of ions
o Hydrophobicity of the molecules
127
Hydrophilic gels
| Gel Chromatography
soluble in aqueous medium
ex. dextran, agarose, polyacrylamide
128
Hydrophobic gels
| Gel Chromatography
soluble in organic solvents
ex. methylatede sephadex, polystyrene beads
129
Clinical use:
| Gel Chromatography
fraction of polysaccharides, NA, proteins, enzymes, isoenzymes
130
Principle: separating and measuring nanograms and pictogram amounts of volatile substance
Gas Chromatography
131
Kinds of GC:
sorbent is solid
w/ a large surface
| Gas Chromatography
Gas-Solid Chromatography
132
Kinds of GC:
sorbent is a
non-volatile liquid
| Gas Chromatography
Gas-Liquid Chromatography
133
Basis of Separation:
| Gas Chromatography
o sample volatility
o rate of diffusion into liquid layer of the column packing
o solubility of sample in the liquid layer
134
Clinical use:
| Gas Chromatography
drug screening and drug analysis
fractionation of steroids, lipids, barbituates, blood alcohol and other toxicologic substances
135
Principle: measures the difference in voltage at a constant
current
Potentiometry
136
relationship between the measured voltage
and the unknown concentration
| Potentiometry
Nerst Equation
137
Principle: measurement of difference in current at a
constant voltage
Polarography
138
relationship between the difference in
current and voltage
| Polarography
Ilkovic Equation
139
Principle: measures the current flow between two nonpolarizable electrodes between a known electrical potential is
established
Conductometry
140
Principle: measures the amount of current that flows when constant voltage is applied the measuring electrode
Amperometry
141
Principle: measures the amount of electricity (coulombs) at a fixed potential
Coulometry
142
1 coloumb =
| Coulometry
1 amper per second
143
number of coulombs consumed can be
directly related to the concentration of the unknown
| Coulometry
Faraday’s Law
144
Principle: Measures the fluorescence or the energy emission that occurs when a certain compound absorbs electromagnetic radiation, become excited and then return to an energy state that is usually higher than their original level
Fluorimetry
145
Emitted light has longer wavelength than the incident/excited light due to the loss of energy during collision
Fluorimetry
146
energy is equal to or lower than the absorbed energy (rarely happens)
| Fluorimetry
Phosphorescence
146
Main problem w/ fluorescence:
| Fluorimetry
Quenching
147
Principle:migration or movement of charged particles in an
electric field
Electrophoresis
148
Factors affecting rate of migration:
| Electrophoresis
1. Net electric charge
2. Size & Shape of Molecule
3. Electric Fluid Strength
149
higher electric charge = faster migration
| Electrophoresis
Net electric charge
150
bigger molecules = slower migration
| Electrophoresis
Size & Shape of Molecule
151
higher ionic strength = slower movement
| Electrophoresis
Electric Fluid Strength
152
more voltage = more movement
| Electrophoresis
Temperature
152
*Problem with increased temp:
| Electrophoresis
oDenaturation of Proteins
oEvaporation of solvent increases ionic strength
153
earliest support media
Disadv: paper is fragile and easily damaged staining of protein
| Electrophoresis
Paper Electrophoresis
153
Nature of Supporting Media:
| Electrophoresis
1. Paper Electrophoresis
2. Starch Gel Electrophoresis-
3. Cellulose Acetate Electrophoresis
4. Agarose Electrophoresis
5. Polyacrylamide Gel Electrophoresis
154
good for large samp
Disadv: fragile and unable to store results permanently
| Electrophoresis
Starch Gel Electrophoresis
155
strip with a clear plastic backing w/ a coating of cellulose acetate particles attached to it
Disadv: becomes brittle when dried
| Electrophoresis
Cellulose Acetate Electrophoresis
156
Disadv: electric neutrality; separation is strictly on the basis of electric charge and uniformity of material size
| Electrophoresis
Agarose Electrophoresis
156
uses protein size as the major factor in the separation process and the net charge of proteins
| Electrophoresis
Polyacrylamide Gel Electrophoresis
157
Clinical Use:
| Electrophoresis
analysis of proteins (serum) that can provide
quick and useful information regarding the presence or absence of disease entities
158
Specimens:
| Electrophoresis
Serum, Urine, Cerebrospinal Fluid
159
# 1.
Advantages:
o rapid results
o increases number of tests performed
o saves time and effort
o eliminates the need for more staff
o economical
o reduces errors in calculation and transcription
o better precision and accuracy
Automation
160
Basic Approaches in Automation:
| Automation
1. CONTINUOUS FLOW ANALYZER
2. DISCRETE ANALYZER
3. CENTRIFUGAL ANALYZER
161
-Sequential analysis
-Uniformity in test performance
| Automation
CONTINUOUS FLOW ANALYZER
162
-Separate analysis
-Most popular and versatile analyzer
| Automation
DISCRETE ANALYZER
163
-Batch analysis
-Centrifugal force moves the reagents and sample to a mixing chamber, into a cuvette, passing a light beam and measuring the absorbance
| Automation
CENTRIFUGAL ANALYZER
