[1S] UNIT 4 Analytical Techniques & Automation Flashcards
1
Q
Four Major Disciplines of Analytic Techniques
A
- Spectrometry
- Luminescence
- Electroanalytic Methods
- Chromatography
2
Q
Is the distance between 2 successive peaks and it is expressed in terms of nanometer (nm)
A
Wavelength
3
Q
<400 nm =
400-700 nm =
>700 nm =
A
UV
Visible
Infrared
4
Q
The longer the wavelength, the ___________ the energy
A
lower
5
Q
The higher the frequency, the _______ the energy
A
higher
6
Q
Planck’s formula
A
E = hv
7
Q
BEER’S LAW
Concentration of the unknown substance is ________ ________ to the absorbed light (absorbance or optical density) and ________ ________ to the amount of transmitted light.
A
directly proportional
inversely proportional
8
Q
T/F: A solution transmits light corresponding in wavelength to its color, and usually absorbs light of wavelengths complementary to its color
A
T
9
Q
Which wavelength would be absorbed strongly by a yellow-colored solution?
a. 450 nm c. 600 nm
b. 585 nm d. 650 nm
A
A
10
Q
A blue-colored solution would show highest transmittance at:
a. 475 nm c. 585 nm
b. 525 nm d. 620 nm
A
A
11
Q
Parts of the Spectrophotometer
A
LEME CPR kya mn yn
12
Q
Parts of the Spectrophotometer
A
LEME CPR kya mn yn
13
Q
LIGHT SOURCE: CONTINUUM SOURCE
Tungsten
A
Infrared & Visible Light
14
Q
LIGHT SOURCE: CONTINUUM SOURCE
Deuterium & Xenon
A
UV
15
Q
LIGHT SOURCE: LINE SOURCE
Mercury-vapor lamps
A
Visible & UV
16
Q
Which of the following light source provides wavelengths of light that fall under the infrared region?
a. Mercury
b. Xenon
c. Deuterium
d. Silicon Carbide
A
D
17
Q
Which of the following light source provides wavelengths of light that fall under the infrared region?
a. Mercury
b. Xenon
c. Deuterium
d. Silicon Carbide
A
D
18
Q
PARTS OF SPECTROPHOTOMETER
Minimizes unwanted stray of light
Prevents entrance of scattered light on the monochromator system
A
Entrance Slit
19
Q
ENTRANCE SLIT
- Any wavelength outside the band transmitted by the monochromator
- Causes absorbance error
A
Stray Light
20
Q
ENTRANCE SLIT
- Limits the maximum absorbance that a spectrophotometer can achieve
- Most common cause of lost linearity at high analyte concentration
A
Stray Light
21
Q
PARTS OF SPECTROPHOTOMETER
It isolates specific or individual wavelength of light
A
Monochromator
22
Q
PARTS OF SPECTROPHOTOMETER
Kinds of Monochromator
A
- Prisms
- Diffraction Gratings
- Filters
- Holographic Gratings
23
Q
PARTS OF SPECTROPHOTOMETER
Controls the width of the light beam (bandpass)
A
Exit Slit
24
Q
PARTS OF SPECTROPHOTOMETER
Furthermore, a bandpass can be described as the ________________.
A
Exit Slit; total range of wavelengths transmitted
25
PARTS OF SPECTROPHOTOMETER
Allows only a narrow fraction of the spectrum to reach the sample cuvette
Exit Slit
26
PARTS OF SPECTROPHOTOMETER
holds the solution whose concentration is to be measured
Cuvette
27
PARTS OF SPECTROPHOTOMETER
Kinds of Cuvette
1. Alumina Silica Glass
2. Quartz Plastic
3. Borosilicate glass
4. Soft glass
28
PARTS OF SPECTROPHOTOMETER: KINDS OF CUVETTE
Capable of withstanding light with wavelength of 350-2000 nm
Alumina Silica Glass
29
PARTS OF SPECTROPHOTOMETER: KINDS OF CUVETTE
Visible-UV
Quartz Plastic
30
PARTS OF SPECTROPHOTOMETER
detects and converts transmitted light into photoelectric energy
Photodetector
31
PARTS OF SPECTROPHOTOMETER
Kinds of Photodetectors
1. Photocell
2. Phototube
3. Photomultiplier tube
4. Photodiode
32
PARTS OF SPECTROPHOTOMETER: PHOTODETECTORS
Most sensitive; used in fluorometric assay
Photomultiplier Tube
33
Compare single beam and double beam
Single Beam = 1 cuvet
Double Beam = 2 cuvets (Sample & Reference)
34
DOUBLE BEAM
2 photodetectors & cuvettes
In Space
35
DOUBLE BEAM
Only 1 photodetector
In Time
36
INSTRUMENTATION
Measures light emitted by one atom burned in a flame
Flame Emission Photometry
37
INSTRUMENTATION PRINCIPLE
Measures the excitation of electrons from lower to higher state
Flame Emission Photometry
38
INSTRUMENTATION PHOTODETECTOR & LIGHT SOURCE
Photo cell
Flame
Flame Emission Photometry
39
INSTRUMENTATION PHOTODETECTOR & LIGHT SOURCE
Photo cell
Flame
Flame Emission Photometry
40
INSTRUMENTATION
Measures light absorbed by one atom in a dissociated by heat
Atomic Absorption Spectrophotometry
41
INSTRUMENTATION
Measures light absorbed by one atom in a dissociated by heat
Atomic Absorption Spectrophotometry
42
INSTRUMENTATION
Most sensitive & specific for electrolytes and trace elements
Atomic Absorption Spectrophotometry
43
INSTRUMENTATION PRINCIPLE
Dissociation of subatomic bonds in electrolytes
Atomic Absorption Spectrophotometry
44
INSTRUMENTATION PHOTODETECTOR & LIGHT SOURCE
Photomultiplier Tube
Hollow Cathode Lamp
Atomic Absorption Spectrophotometry
45
When measuring calcium by atomic absorption spectrophotometry, which is required?
a. An organic extraction reagent to deconjugate calcium from protein
b. An internal standard
c. A magnesium chelator
d. Lanthanum oxide to chelate phosphates
D
46
INSTRUMENTATION PRINCIPLE
The unknown sample is made to react with a known solution in the presence of an indicator
Volumetric (Titremetric)
47
INSTRUMENTATION: VOLUMETRIC
For determination of fluoride concentration in the bloodstream
Schales and Schales Method
48
INSTRUMENTATION: VOLUMETRIC
For determination of fluorife concentration in the bloodstream
Schales and Schales Method
49
INSTRUMENTATION: VOLUMETRIC
Chelates calcium
EDTA Titration Method
50
INSTRUMENTATION PRINCIPLE
- It determines the amount of scattered light by a particulate matter suspended in a turbid solution
- Measures protein in a sample
Nephelometry
51
INSTRUMENTATION: NEPHELOMETRY
Light Scattering depends on the following factors?
a. Particle Size
b. Wavelength
52
INSTRUMENTATION
➢ Measures the amount of Antigen-Antibody Complexes.
➢ Measures the angle at 15-90 degrees.
Nephelometry
53
Used to descrive a molecule that invoke an immune response
Antigens
54
Most anti antigens to least (d k alam bt gnyn pero yn nsa notes k bhala kau)
1. Proteins
2. Polysaccharides
3. Nucleic Acids & Lipids
55
1. Direct neutralization
2. Opsonization
3. Complement activation
4. Somatization
5. Control of inflammatory response
NEPHELOMETRY: Antibody Molecule
56
INSTRUMENTATION PRINCIPLE
Measures reduction (not specific protein) in light transmission by one particle formation
Turbidimetry
57
This is the process of separating the charged constituents of a sample by means of an electrical current
Electrophoresis
58
ELECTROPHORESIS
Migration of charged macromolecules in the presence of an electrical power through a porous support:
a. Paper
b. Cellulose acetate
c. Agarose gel
d. Polysaccharide
Zone Electrophoresis
59
ELECTROPHORESIS
Has a net charge that can be either positive or negative depending on pH conditions
Amphoteric
60
ELECTROPHORESIS
T/F: Amphoteric Electrophoresis is always done in acidic solution (2.0) to become negatively charged
F; alkaline solution (9.0)
61
ELECTROPHORESIS
Movement of buffer ions & solvent relatives to the fixed support
Electroendosmosis/Endosmosis
62
ELECTROPHORESIS
Migration of small charged ions
Iontophoresis
63
5 Components of Electrophoresis
1. The driving force
2. The support medium
3. The buffer
4. The sample
5. The detecting system
64
3 Electrophoresis Support Media
1. Cellulose acetate
2. Agarose gel
3. Polyacrylamide gel
65
6 Electrophoresis Procedures (Proteins)
1. Loading of patient sample
2. Electrophoretic migration
3. Wash and Fix
4. Staining
5. Visualization (Qualitative Result)
6. Quantification
66
5 Electrophoresis Fractions of Separated Proteins
1. Albumin
2. Alpha1
3. Alpha2
4. Beta
5. Gamma globulins
67
7 Electrophoresis Stains for Visualization of Bands
1. Amido Black
2. Ponceau S
3. Oil Red O
4. Sudan Black
5. Fat Red 7B
6. Coomassie Blue
7. Gold/Silver Stain
68
ELECTROPHORESIS STAINS FOR VISUALIZATION OF BANDS
For plasma proteins
Amido Black
69
ELECTROPHORESIS STAINS FOR VISUALIZATION OF BANDS
Proteins in very minute amounts
Gold/Silver Stain
70
What dye maybe used for staining protein
bands following electrophoresis?
a. Fat red 7B
b. Sudan black B
c. Ponceau S
d. Oil red O
c. Ponceau S
71
Which of the following reagents can be used to measure protein present in the cerebrospinal fluid?
a. Biuret
b. Coomassie brilliant blue
c. Ponceau S
d. Bromcresol green
b. Coomassie brilliant blue
72
INSTRUMENTATION PRINCIPLE
Involves the separation of soluble materials in a solution by specific chemical and physical differences through:
- Rate of diffusion
- Solubility
- Size
- Ionic charges
Chromatography
73
CHROMATOGRAPHY
Diffusion of molecules in 2D-plane system
Planar Form Chromatography
74
PLANAR FORM CHROMATOGRAPHY
Fractionation of sugar and amino acids through whattaman paper sorbent
Paper Chromatography
75
PLANAR FORM CHROMATOGRAPHY
Used for TDM to separate drug molecules through plastic plates sorbent
Thin Layer Chromatography
76
CHROMATOGRAPHY
Diffusion of analytes in 3D-multidirectional system
Column Form Chromatography
77
COLUMN FORM CHROMATOGRAPHY
Elution of volatile compounds based on boiling point, used to separate steroids, lipids, alcohols
Gas Chromatography
78
COLUMN FORM CHROMATOGRAPHY: GAS
Differences in absorption of gases sat solid phase surfaces
Gas Solid Chromatography
79
COLUMN FORM CHROMATOGRAPHY: GAS
Differences in solute partitioning between gaseous mobile phase vs liquid stationary phase
Gas Liquid Chromatography
80
COLUMN FORM CHROMATOGRAPHY
Distribution of solute betweem liquid mobile phase vs liquid stationary phase
Liquid Chromatography
81
COLUMN FORM CHROMATOGRAPHY: LIQUID
Most widely used, uses pressure for fast separation, fractionation of drugs, hormones & Hgb variants
HPLC
82
COLUMN FORM CHROMATOGRAPHY: LIQUID
Separate non-volatile substances in human body fluids, complementary method to GC-MS as it is used to confirm positive results from screening illicit drugs
LC-MS
83
Fragmentation and ionization of molecules using suitable source of energy (ex: CT scan)
Mass Spectroscopy
84
- Advantage: Requires smaller sample volume
- Disadvantage: Destructive
Mass Spectroscopy
85
Determine the structure of organic compound (ex: MRI)
Nuclear Magnetic Resonance Spectroscopy
86
- Advantage: Non-destructive
- Disadvantage: Requires larger sample volume
Nuclear Magnetic Resonance Spectroscopy
87
SEPARATION TECHNIQUES
Migration is based on electrical charge
Electrophoresis
88
SEPARATION TECHNIQUES
Migration is based on physical/chemical properties
Chromatography
89
SEPARATION TECHNIQUES
Migration is through a pH gradient
Isoelectric Focusing
90
SEPARATION TECHNIQUES
Migration is through an electro-osmosis flow
Capillary Electrophoresis
91
INSTRUMENTATION PRINCIPLE
Amount of light emitted by a molecule after excitation by electromagnetic radiation
Fluorometry
92
INSTRUMENTATION
- Measures amount of light intensity present over a dark background
- Utilizes 2 monochromators
Fluorometry
93
INSTRUMENTATION
Advantage: More specific & 1000x more sensitive than spectro
Fluorometry
94
INSTRUMENTATION
Light source of Fluorometry
Mercury
Xenon
UV Lights
95
INSTRUMENTATION
Photodetector of Fluorometry
PMT / Phototube
96
FLUOROMETRY
T/F: Affected by photobleaching - pH, temp, UV, and chemical changes
T; quenching & photobleaching r same bananas
97
FLUOROMETRY
T/F: Quenching is the reduction or limitation of a particle in an excited state
T
98
↑ Light Exposure = _ Fluoroscence
↑
99
↑ Unwanted light exposure = _ Fluoroscence
↓
100
↑Temperature = _ Fluoroscence
↓
101
↑ Quenching = _ Fluoroscence
↓
102
↑ Absorbing analytes = _ Fluoroscence
↓
103
Detection systems used in real time PCR are based on:
a. radioactivity
b. chemiluminescence
c. fluorescence
d. colorimetry
c. fluorescence
104
INSTRUMENTATION PRINCIPLE
Measurement of luminescence produced by chemical reaction producing light emission
Chemiluminescence
105
INSTRUMENTATION PRINCIPLE
Measurement of luminescence produced by chemical reaction producing light emission
Chemiluminescence
106
INSTRUMENTATION
Measures amount of light emission based on chemical or electrochemical reaction
Chemiluminescence
107
INSTRUMENTATION
More sensitive than fluorometry and spectrophotometry
Chemiluminescence
108
INSTRUMENTATION
More sensitive than fluorometry and spectrophotometry
Chemiluminescence
109
Which of the following components is not needed in a chemiluminescent immunoassay analyzer?
a. Source lamp
b. Monochromator
c. Photodetector
d. Wash station
a. Source lamp
110
INSTRUMENTATION PRINCIPLE
● changes in colligative property of solutions that occur due to variations in particle concentrations
● Measurement of the osmolarity of an aqueous solution
○ (Ex.) osmotic particles: Glucose, BUN, Sodium
Osmometer
111
Increased osmolarity = Increased osmotic pressure, boiling point = _______ vapor pressure, freezing point
Decreased
112
ELECTROCHEMISTRY
● Measurement of electrical potential due to the activity of free ions
● Change of voltage indicates analyte activity (potential means voltage)
POTENTIOMETRY
113
ELECTROCHEMISTRY
● Reference electrodes
○ Calomel
○ Silver-Silver Chloride
○ H+ Electrode
● Measures: pH and pCO2
● Relies on the concept of Nerst Equation
POTENTIOMETRY
114
ELECTROCHEMISTRY
● Electrochemical transducer capable of responding to one given ion
● Very sensitive and selective, yet non-specific
● A type of potentiometer
ION SELECTIVE ELECTRODE
115
ION SELECTIVE ELECTRODE MEMBRANES
For measuring sodium
Glass aluminum silicate
116
ION SELECTIVE ELECTRODE MEMBRANES
For potassium measurement
Valinomycin gel
117
ION SELECTIVE ELECTRODE MEMBRANES
For calcium and lithium
Organic liquid
118
● Direct ISE: uses ______ sample
● Indirect ISE: uses _______ sample
● diluted sample
● undiluted sample
118
ELECTROCHEMISTRY
● Measures: chloride test (CSF, serum, sweat)
● Governed by Faraday’s Law
○ For monitoring cystic fibrosis
COULOMETRY
119
ELECTROCHEMISTRY
● Measurement of the amount of electricity at fixed potential coulombs
● Electrochemical titration in which the titrant is electrochemically generated and the end point is detected by amperometry
COULOMETRY
120
ELECTROCHEMISTRY
Measures: pO2, glucose, chloride, peroxidases
AMPEROMETRY
121
ELECTROCHEMISTRY
● Current flow produced by oxidation reaction
AMPEROMETRY
122
ELECTROCHEMISTRY
● Measurement of differences in current at a constant voltage (Clarke’s Polarographic Electrode)
● A type of amperometry, uses Ilkovic Equation
POLAROGRAPHY
123
ELECTROCHEMISTRY
● Measurement of current after a potential is applied to an electrochemical cell
● Allows sample to be pre-concentrated, thus minimal analyte
VOLTAMMETRY
124
ELECTROCHEMISTRY
Anodic stripping voltammetry: specifically utilized for lead and iron studies
VOLTAMMETRY
125
AUTOMATION IN THE CLINICAL LABORATORY
● Samples flow through a common vessel or pathway
● Liquids are pumped through a system of continuous tubing
CONTINUOUS FLOW ANALYZER
126
AUTOMATION IN THE CLINICAL LABORATORY
Air bubbles serve as separating and cleaning media
CONTINUOUS FLOW ANALYZER
127
AUTOMATION IN THE CLINICAL LABORATORY
Allow random access or STAT capabilities
DISCRETE ANALYZER
127
AUTOMATION IN THE CLINICAL LABORATORY
● Each sample-reagent mixture is handled in its own reaction vessel
● Employs a variety of syringe pipettes to aspirate and dispense samples (2-6 uL) and reagents
DISCRETE ANALYZER
128
AUTOMATION IN THE CLINICAL LABORATORY
● Uses the force generated by centrifugation to transfer specimen and reagents
● Major advantage: Batch analysis
CENTRIFUGAL ANALYZER
129
GLASSWARES
- Up to 515 degrees celcius
- Most common (heating & sterilization)
Borosilicate Glass (Pyrex, Kimax)
130
GLASSWARES
- High thermal resistance
- Low alkali content (free from magnesium lime)
Borosilicate Glass (Pyrex, Kimax)
131
GLASSWARES
- Less thermal resistance than borosilicate
- High alkali resistance
Boron-free Glass (Soft Glass)
132
GLASSWARES
- 6x stronger than borosilicate
- Strengthened chemically than thermally
Corex (Corning, Alumina-silicate Glass)
133
GLASSWARES
- Up to 900C
- High thermal-drastic heat shock resistance
- Extremely chemical (acid, alkali) resistant
Vycor (Corning)
134
GLASSWARES
- Poor thermal resistance
- Disposable
Flint Glass (Soda-lime Glass)
135
GLASS PIPET ACCDNG TO CALIB DESIGN
Holds volume but don’t dispense exact amount
TC
136
GLASS PIPET ACCDNG TO CALIB DESIGN
Delivers exact amount
TD
137
GLASS PIPET ACCDNG TO DRAINAGE CHARAC
Etched ring, need to blow last drop to get exact volume
Blow-out
138
GLASS PIPET ACCDNG TO DRAINAGE CHARAC
Etched ring, gravity auto-drains the liquid
Self-draining
139
NEPHELOMETRY
If a particular analyte has a larger size than the wavelength of light, it will scatter the light in a forward direction in an angle of 90 degrees or lower
Mie scatter
140
INSTRUMENTATION
Light source of Nephelometry
Laser
