U3 LAB: INSTRUMENTATION Flashcards
1
Q
Four Major Disciplines of Analytic Techniques
A
- Spectrometry
- Luminescence
- Electroanalytic methods
- Chromatography
2
Q
under Spectrophotometry
A
- Spectrophotometry
- Flame Emission Spectrophotometry
- Atomic Absorption
3
Q
Measures intensity of light or light transmitted
A
Spectrophotometry
4
Q
Spectrophotometry
directly proportional to concentration
A
Absorbance
5
Q
Spectrophotometry
inversely proportional to concentration
A
Transmittance
6
Q
Measures intensity of light after an ion is burned (light of a single atom)
A
Flame Emission Spectrophotometry
7
Q
Principle of Flame Emission Spectrophotometry
A
excitation of electrons from lower to higher energy
8
Q
Measure of light absorbed after ions are dissociated by heat
A
Atomic Absorption
9
Q
Atomic Absorption is used to measure?
A
trace elements (Ca, Mg)
10
Q
Measures the light produced when ions are excited to unexcited
A
Fluorometer
11
Q
Luminescence
Material absorbs at high energy but short wavelength, emits light at lower energy (visible light)
A
Fluorescence
12
Q
Luminescence
Measures analyte produced in the reaction vessels
A
Chemiluminescence
13
Q
Chemiluminescence produces?
A
electromagnetic radiation of UV, visible, infrared
14
Q
Luminescence
Chemical yields electronically excited intermediate/product responsible for the emission
A
Chemiluminescence
15
Q
Luminescence
for proteinous analytes
A
Nephelometry
16
Q
Luminescence
measures the light scattered by a particulate matter suspended in a solution
A
Nephelometry
17
Q
Luminescence
Most common example of Nephelometry
A
measurement of antigen-antibody complexes (turbidimetry)
18
Q
Separation of molecules by their molecular weight
A
Electrophoresis
19
Q
This refers to separation techniques for soluble components in a solution by specific differences in physical or chemical characteristics.
A
Chromatography
20
Q
Separation of compounds that are naturally volatile or chemically converted to a volatile form
A
Gas Chromatography
21
Q
Beer Lambert’s Law
A
concentration of analyte is directly proportional to absorbance
22
Q
The darker the sample?
A
the higher concentration, intensity, absorbance
23
Q
<400nm
A
Ultraviolet (UV)
24
Q
400-700nm
A
Visible Light
25
>700nm
infrared
26
750-1600nm
mid IR / fingerprint region
27
longer wavelength, _____ energy and frequency
lower
28
shorter wavelength, _________ energy and frequency
higher
29
Planck's Formula
E = hv
30
Planck's Formula
E
energy of photon in Joules
31
Planck's Formula
h (constant)
6.626 x 10-34
32
Planck's Formula
v
frequency of electromagnetic radiation
33
Beer Lambert's Law
What is inversely proportional?
Transmittance and absorbance
34
T/F: A solution transmits light corresponding in wavelength to its color, and usually absorbs light of wavelengths complementary to its color.
True
35
This minimizes unwanted stray light.
Entrance slit
36
This eliminates unwanted wavelengths.
Monochromater
37
Holds the solution
Cuvette
38
Two types of Light Source
- Continuum Source
- Line Source
39
Continuum Source
- Tungsten
- Deuterium
- Xenon
40
Continuum Source
Most common for visible infrared light sources
Tunsgten
41
Continuum Source
Routinely used for UV based light sources
Deuterium
42
Continuum Source
Used in most spectrofluorometers
Xenon
43
under Line source
Mercury-vapor lamps
44
Light under infrared region
Silicon Carbide
45
Stray light is considered as?
- any wavelength outside band
- causes absorbance error
- limits the maximum absorbance
- most common cause of lost linearity
46
Stray light can come from?
deteriorating light source
47
Kinds of Monochromator
- Prisms
- Diffraction Gratings
- Filters
- Holographic Gratings
48
Kinds of Monochromator
made up of glass
Prisms
49
Kinds of Monochromator
has grooves
Diffraction Gratings
50
This controls the width of the light beam.
Exit slit
51
Described as the total range of wavelengths transmitted
bandpass
52
Cuvette is also known as?
Analytical / Absorption / Sample Cell
53
Kinds of Cuvette
- Alumina Silica Glass
- Quartz Plastic
- Borosilicate glass
- Soft glass
54
Kinds of Cuvette
most common, wide range absorbance
Alumina Silica Glass
55
Kinds of Cuvette
Alumina Silica Glass has an absorbance range of?
350 - 2000nm
56
Kinds of Cuvette
measurement of solution requiring a UV and visible spectra
Quartz Plastic
57
Notes to remember for cuvette
1.) discard cuvettes with scratches (cause interferences)
2.) silica cuvettes transmit light effectively at wavelengths above 220nm
3.) prolonged alkaline solution may cause cuvette to corrode or dissolve
58
This detects and converts transmitted light into photoelectric energy.
Photodetector
59
Kinds of Photodetector
- Photocell
- Phototube
- Photomultiplier tube
- Photodiode
60
Kinds of Photodetector
most simple
Photocell
61
Kinds of Photodetector
most sensitive as it amplifies light first before converting to electricity
Photomultiplier tube
62
This splits monochromatic light into 2
Beam splitters
63
Double beam in space
2 photodetectors
64
Double beam in time
1 photodetector
65
FEP
Principle
excitation of electron from low energy to high energy
66
FEP
Photodetector
photocell
67
FEP
Light source
flame (also serves as cuvette)
68
FEP
Sodium
yellow-orange
69
FEP
Potassium
pink
70
FEP
Calcium
orange
71
FEP
Magnesium
bright white
72
FEP
Copper I
blue
73
FEP
Copper II
green
74
FEP
Lithium
red
75
FEP
Cesium
purple
76
FEP
known as internal standards and collects variation in flames
Lithium and Cesium
77
Most specific and most sensitive spectrophotometry
Atomic Absorption
78
AAS
Principle
element is not excited but merely dissociates from its chemical bond and placed in an unionized gram stain
79
AAS
Photodetector
Photomultiplier tube (measures trace elements)
80
AAS
Light source
Hollow cathode lamp
81
Volumetric (Titrimetric)
Principle
unknown sample is made to react with a known solution in the presence of an indicator
82
Volumetric (Titrimetric)
Examples
- Schales and Schales
- EDTA Titration method
83
Volumetric (Titrimetric)
Schales and Schales
for chloride
84
Volumetric (Titrimetric)
EDTA Titration
for calcium
85
Nephelometry
Principle
determines amount of scattered light by a particulate matter suspended in a turbid solution
86
Nephelometry
Light scattering depends on:
- particle size
- wavelength
87
Nephelometry
Angle
15 to 90 degrees
88
Nature of antigen
provokes immune response
89
Detection methods of the antibody molecule
- Direct neutralization
- Opsonization
- Complement activation
- Somatization
- Control of inflammatory response
90
Phenomenon of forward scatter
Mie scatter
91
Mie scatter occurs because most complexes have a diameter of around?
250-1500nm
92
Wavelengths in Mie scatter
320 to 650nm
93
Light source of nephelometry
- laser (most common)
- light amplification (stimulated emission of radiation)
- Tungsten Iodide lamp
94
Turbidimetry
Principle
measures reduction in light transmission by one particle formation
95
measure of blocked light or light reduced in large particles by a particulate matter in a solution
Turbidimetry
96
Clinical applications of Turbidimetry
- CSF
- urine
97
Phenomenon in Turbidimetry
Raleigh scatter
98
This refers to scattered light in any directions, particles are smaller than wavelengths of light.
Raleigh scatter
99
T/F: Raleigh scatter will scatted light in many directions, but not equally forward and backward.
False
equal lang
100
separate DNA and RNA based on size and electrical charge
Electrophoresis
101
separating charged constitutents of a sample by means of an electrical current
Electrophoresis
102
Migration of charged macromolecules in presence of electrical power through porous support
Zone electrophoresis
103
Porous support for electrophoresis
- Paper
- Cellulose acetate (densitometry)
- Agarose gel (DNA, RNA, proteins)
104
has a net charge that can be either positive or negative depending on pH conditions
Amphoteric
105
movement of buffer ions and solvent relatives to the fixed support
Electroendosmosis / Endosmosis
106
migration of small charged ions
Iontrophoresis
107
Five components of Electrophoresis
1. driving force (electrical power)
2. support medium (gel, cellulose paper)
3. buffer
4. sample
5. detecting system
108
Detecting system for DNA
UV Transilluminator
109
Detecting system for Proteins
Densitometry
110
Support media for Electrophoresis
- Cellulose acetate
- Agarose gel
- Polyacrylamide gel
111
This separates serum into 5 bands.
Cellulose acetate
112
Support media for DNA, separates 10-15 bands, predominant component of agar
Agarose gel
113
For protein, separates based on charge and molecular size, 30 fractions, neurotoxic, used to study isoenzymes
Polyacrylamide gel
114
This is lighter than the molecule of interest.
Tracking dye
115
T/F: The brighter the band, the higher the protein is in electrophoresis
True
116
Procedure for Electrophoresis
1. Loading of sample
2. Electrophoretic migration
3. Wash and fix
4. Staining
5. Visualization
6. Quantification
117
5 Bands of Separated Proteins
- Albumin
- Alpha 1
- Alpha 2
- Beta
- Gamma
118
Stains for Proteins
- Amido Black
- Ponceau S
- Oil Red O
119
Stains for Lipids
- Sudan Black
- Fat Red 7B
120
Other stains
- Coomassie Blue
- Gold/Silver Stain
121
Stain that is very sensitive to nanograms of proteins
Gold/Silver Stain
122
Reagent to measure protein in CSF
Coomassie brilliant blue
123
Elution of volatile compounds based on boiling point, used to separate steroids, lipids, alcohols
Gas Chromatography
124
Fragmentation and ionization of molecules
Mass Spectroscopy
125
GC-MS is gold standard for?
drug testing
126
MS/MS is gold standard for?
newborn screening
127
used to determine structure of organic compound (e.g. MRI)
Nuclear Magnetic Resonance Spectroscopy
128
Migration based on electrical charge
Electrophoresis
129
Migration based on physical/chemical properties
Chromatography
130
Migration is through a pH gradient
Isoelectric focusing
131
Migration is through electro-osmosis flow
Capillary Electrophoresis
132
Measures light from excited to unexcited
Fluorometry
133
Fluorometry
Primary filter
UV light
134
Fluorometry
Secondary filter
Visible light
135
Detection systems in PCR is based on?
fluorescence
136
Chemiluminescence
Principle
measurement of luminescence produced by chemical reaction producing light emission
