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Clinical Chemistry Lecture 2 > Analytical Methods > Flashcards

Analytical Methods Flashcards

(182 cards)

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0
Q

When is light emitted in fluorometry?

A

When the molecule has returned to the more stable ground state

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1
Q

Principle of mass spectrometry

A

Based on fragmentation and ionization of molecules using a suitable source of energy

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2
Q

4 basic disciplines of analytic methods

A

1 Spectrometry
2 Luminescence
3 Electroanalytic methods
4 Chromatography

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3
Q

Spectrometry

A

Spectrophotometry
Atomic absorption
Mass spectrometry

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4
Q

Type of optical methods

A

Absorption
Emission
Polarization
Scattering

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5
Q

Examples of emission methods

A

Flame emission spectrophotometry
Fluorescence correlation spectroscopy
Fluorescence energy transfer spectroscopy
Fluorometry
Luminometry (light emission from a bioluminescent, chemiluminescent, or electrochemiluminescent reaction)
Phosphorimetry
Time-resolved fluorometry

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6
Q

Luminescence

A

Fluorescence
Chemiluminescence
Nephelometry

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7
Q

Examples of polarization methods

A

Fluorescence polarization spectroscopy

Polarimetry

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9
Q

Examples of scattering methods

A

Nephelometry

Turbidimetry

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10
Q

It describes the radiant energy with wavelengths visible to the human eye

A

Light

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11
Q

Short wavelength

A

Gamma rays

X-rays

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12
Q

Longer wavelength

A

Radio

Microwave

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13
Q

400 nm wavelength

A

Violet

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14
Q

700 nm wavelength

A

Red

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15
Q

Human eye

A

380-750 nm

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16
Q

Measures shorter (uv) or longer (infrared) wavelength

A

Photometric apparatus

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17
Q

Short wavelength, high frequency

A

High gamma rays

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18
Q

T or F. When light is absorbed, it is transmitted.

A

F. When light is not absorbed, it is transmitted.

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19
Q

Used to select the incident wavelength

A

Filters (photometers)

Prisms or gratings (spectrometers)

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20
Q

UV at 200-380 nm

A

Near UV

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21
Q

Examples of absorption methods

A 
Atomic absorption
Densitometry
Fourier transform infrared spectroscopy
Photometry
Spectrophotometry
Reflectance photometry
X-ray spectroscopy
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22
Q

UV at < 220 nm

A

Far UV

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23
Q

Silica used to make cuvets transmits light effectively at wavelengths _______

A

> /= 220 nm

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24
Q

Principle of spectrophotometry

A

Measurement of the light transmitted by a solution to determine concentration of light-absorbing substances in solution

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25
May either be single-beam or double-beam
Spectrophotometer
26
Instruments used in spectrophotometry
Filter photometers | Spectrophotometers
27
What is a single-beam spectrophotometer?
It makes one measurement at a time at one specified wavelength.
28
What does Beer's law state?
The concentration of a substance is directly proportional to the light absorbed or inversely proportional to the logarithm of the transmitted light.
29
Light source of a spectrophotometer
Incident light
30
It determines the color of light seen by the eye
Wavelength of light
31
Types of incident light used by a spectrophotometer
``` 1 Continuum Deuterium (< 300 nm) Tungsten (400-800 nm) Xenon Change in intensity Adapt ``` 2 Light Cathode lamp Does not adapt
32
Selects wavelength in a spectrophotometer
Monochromator
33
Types of monochromator
Colored glass filters Interference filters Prism Diffraction grating
34
What is a double-beam spectrophotometer?
It splits monochromatic light into two components and records absorbance of a sample directly.
35
Most common material used in making cuvets
Silicate
36
Affect the results when present in the sample holder
Scratch | Alkaline
37
Types of sample holder
Square or round Glass Quartz
38
Parts of a spectrophotometer
``` Light source Collimator Monochromator Slit Sample holder Photodetector Read-out system ```
38
Range detected by glass cuvets
Visible range
38
Detected by quartz cuvets
UV Radiation
39
Types of read-out system
Moving needle | Digital display of output
40
Function of a collimator
Limits stray light
43
Chromatography
Gas Liquid Thin layer
44
Function of an external slit
Limits the band pass
45
Examples of photodetectors
Photocell Phototube Photomultiplier Photodiode
45
Applications of atomic absorption spectrophotometry
Electrolytes (Na, K, Ca, H, Cl) | Dissolved gases
46
It converts transmitted radiant energy into an equivalent amount of electrical energy
Photodetector
46
Light source of atomic absorption spectrophotometers
Hollow cathode lamp
47
Function of beam choppers of atomic absorption spectrophotometers
Used to modulate the light source
48
It converts ions to atoms in atomic absorption spectrophotometry
Chopper
49
Special application of atomic absorption spectrophotometry
Detects small elements when concentration is too low
52
It excites a molecule at the ground state Eo lebel to a higher excited energy level E1
Light absorption
53
Requirement of mass spectrometry before a compound can be detected and quantified
Must be isolated by another method (GC or HPLC)
53
Applications of Gas Chromatography-Mass Spectrometry
Gold standard for drug testing | Proteomics
54
Classification methods
Separation Qualitative Quantitative
55
Major steps in mass spectrometry
1 Conversion of parent molecule into ions 2 Separation of the ions by mass/charge ratio 3 Measurements of current produced when the ions strike a transducer
56
Principle of fluorometry
Measures the amount of light emitted by a molecule after excitation by electromagnetic radiation
58
Electroanalytic methods
Electrophoresis Potentiometry Amperometry Voltammetry
58
Vibrational energy losses
Collisions | Heat losses
59
These molecules can fluoresce
Organic molecules with conjugated double bonds
60
Components of a fluorometer
Light source Monochromator Detector Read-out system
61
Light sources of fluorometers
Mercury arc discharge lamp | Xenon arc tube
62
Forward light scatter nephelometry
Rayleigh-Debye type
63
Examples of monochromators of fluorometers
Diffraction grating | Filter
64
Examples of detectors used in fluorometry
Phototube | Photomultiplier tube
66
Principle of atomic absorption spectrophotometer
Measures the absorption of light of a unique wavelength by atoms in the ground state
67
Types of monochromator used in fluorometry
Primary or grating filter | Secondary filter
68
It allows passage of light of the proper wavelength for absorption of molecule
Primary filter or grating
69
It transmits light of the specific wavelength emitted by the sample
Secondary monochromator
70
Factors that affect fluorescence
pH changes Temperature Length of time of exposure Concentration
71
Clinical application of fluorometry
Measurement of porphyrins, magnesium, calcium, and catecholamines
72
Application of chemiluminescence
Immunoassays
73
Advantages of chemiluminescence
Subpicomolar detection limits Speed Ease of use Simple instrumentation
74
Disadvantage of chemiluminescence
Impurities can cause background signal that degrades sensitivity and specificity
75
Examples of photodetectors used in chemiluminescence
Photomultiplier tube | Luminometer
76
It is taken as the signal in chemiluminescence
Integral of the entire peak
77
Principle of chemiluminescence
The chemical reaction yields an electronically excited compound that emits light as it returns to its ground state or that transfers its energy to another compound which then produces emission
78
Organic compounds in chemiluminescence
Luminol Acridium esters Dioxetanes by oxidants hydrogen peroxide, hypochlorite or oxygen
79
It involves oxidation of an organic compound characterized by a rapid increase in intensity of emitted light followed by a gradual decay
Chemiluminescence
80
Application of nephelometry
Measurement of Ag-Ab reactions
81
It comes from the species
Chemiluminescence
82
Produced as part of the chemical energy generated and decay to a ground state with the emission of photons
Excited intermediates
83
No excitation radiation is required and monochromators are not needed
Chemiluminescence
84
Emitted radiation is measured with a photomultiplier tube and the signal is related to the analyte concentration
Chemiluminescence
85
Components of a nephelometer
``` Light source Collimator Monochromator Simple cuvet Stray light trap Photodetector ```
86
Principle of nephelometry
Measurement of the light scattered by a particulate solution
87
It detects light that is scattered at various angles and the signal that the scattered light yields is amplified
Nephelometry
88
Dependent on wavelength of incident light and particle size
Light scattering
89
Wavelength if light > particle diameter
Symmetrical
90
Wavelength of light < particle diameter
Forward
91
Wavelength of light = particle diameter
More forward
92
Diameter of most Ag-Ab complexes
250-1500 nm
92
Wavelengths used for most Ag-Ab complexes
320-650 nm
93
Sorbent of paper chromatography
Whatman paper
94
Principle of turbidimetry
Measures reduction in light transmission due to particle formation
95
It detects light transmitted in the forward direction
Turbidimetry
96
It is dependent on the specimen concentration and particle size
Amount of light absorbed by a suspension of particles
97
Instruments used in turbidimetry to measure solutions for quanitification
Visible photometers | Visible spectrophotometers
98
Applications of turbidimetry
Protein measurement in CSF and urine Detection of bacterial growth in broth cultures Measurement of antibiotic sensitivities Detection of clot formation
99
Principle of electrophoresis
Separation of charged compounds based on their electrical charge
100
Components of electrophoresis instrument
``` Electrical power Support medium Buffer Sample Detecting medium ```
101
Factors affecting rate of migration in electrophoresis
``` Net electric charge of the molecule Size and shape of the molecule Electric field strength Nature of the supporting medium Temperature of operation ```
102
Produced by the flow of ions when a voltage is applied to a salt solution
Electrical current
103
Supporting media used in electrophoresis
``` Paper electrophoresis Starch gel Cellulose acetate* Agarose gel* Polyacrylamide gel* ```
104
Separates by surface charge and molecular size
Starch gel
105
Separates by molecular size
Cellulose acetate
106
Separates by electrical charge and does not bind protein
Agarose gel
107
Neutral supporting media
Agarose gel | Polyacrylamide gel
108
Used to study isoenzymes
Polyacrylamide gel
109
Neutral; separates on the basis of charge and molecular size
Polyacrylamide gel
110
Stains for visualization of fractions in electrophoresis
``` Amido black* Ponceau S* Oil red O Sudan black* Fat red 7B* Coomassie blue Gold/silver stain ```
111
It measures the absorbance of the stain on a support medium
Densitometer
112
Components of a densitometer
Light source Monochromator Optical system Photodetector
113
Principle of densitometry
Signals detected by the photodetector are related to the absorbance of the sample stain on the support, which is proportional to the specimen concentration
114
It is a modification of electrophoresis
Isoelectric focusing
114
Principle of isoelectric focusing
Charged proteins migrate through a support medium that has a continuous pH gradient
115
Applications of isoelectric focusing
Detects oligoclonal immunoglobulin bands in CSF | Detects isoenzymes of CK, ACP, ALP in serum
116
It follows the Nernst equation
Potentiometry
117
Principle of potentiometry
Concentrations of ions in a solution can be calculated from the measured potential difference between 2 electrodes (reference and indicator electrode)
118
What does potentiometry measure?
Electrical potential due to the activity of free ions (change in voltage indicates activity of each analyte) Differences in voltage (potential) at a constant current
119
What is amperometry?
Measurement of the current flow produced by an oxidation-reduction reaction
120
Applications of amperometry
Determination of pO2, chloride, and peroxidase
121
Advantages of voltammetry
Sensitivity and capability for multi-element measurements (most important) Consumes minimal analyte
122
Method in which a potential is applied to an electrochemical cell and the resulting current is measured
Voltammetry
123
Measures heavy metals like lead
Anodic stripping voltammetry
124
Application of coulometry
Measurement of chloride ion in serum, plasma, CSF, and sweat samples
125
Faraday's law
Q=It=znF Where z= the number of electrons involved in the reaction n= the number of moles of analyte in the sample F= Faraday's constant (96,485 C/mol of electrons)
127
What is chromatography?
A separation method based on different interactions of the specimen compounds with the mobile phase and with the stationary phase as the compounds travel through a support medium
128
2 forms of chromatography
Planar | Column
129
Examples of planar chromatography
Paper | Thin layer
130
Examples of column chromatography
Gas | Liquid
131
Sorbent of thin layer chromatography
Thin plastic plates impregnated with a thin layer of silica gel or alumina
132
Application of thin layer chromatography
Drug screening
133
Advantage of isoelectric focusing
It can resolve mixtures of proteins
134
Application of paper chromatography
Fraction of sugar and amino acid
135
It measures the quantity of electricity (in coulombs) needed to convert an analyte to a different oxidation state
Coulometry
136
Application of gas chromatography
Separation of steroids, barbiturates, blood, alcohol, lipids
137
Useful for compounds that are naturally volatile or can be easily converted into a volatile form
Gas chromatography
138
Types of stationary phases in gas chromatography
Gas-solid | Gas-liquid
139
Separation occurs by differences in absorption at the solid phase surfaces
Gas-solid chromatography
140
Separation occurs by differences in solute partitioning between gaseous mobile phase and liquid stationary phase
Gas-liquid cheomatography
141
Most widely used liquid chromatography
HPLC
142
Based on the distribution of solutes between a liquid mobile phase and a stationary phase
Liquid chromatography
143
Advantages of liquid chromatography over gas chromatography
1 No need for chemical derivatization of organic compounds 2 Use of lower temperature for separation 3 Easy recovery of a sample
144
Bases of separation in chromatography
``` 1 rate of diffusion 2 solubility of solute 3 nature of solvent 4 sample volatility/solubility 5 distribution between 2 liquid phases 6 molecular size 7 hydrophobicity of the molecule 8 ionic attraction 9 differential distribution 10 selective separation of substances 11 differences in absorption and desorption of solutes ```
145
Separation mechanisms in liquid chromatography
``` Gel or molecular sieve Ion exchange Partition Affinity Adsorption ```
146
Separates molecules based on differences in size and shape
Gel or molecular sieve chromatography
147
Uses immobilized biochemical ligands as the stationary phase to separate a few solutes from other unretained solutes
Affinity chromatography
148
Other term for hydrophilic gel
Gel filtration
149
Application of gel filtration
For separation of enzymes, antibodies, proteins
150
Examples of hydrophilic gel
Dextran and agarose
151
Exchange of sample ions and mobile-phase ions with the charged group of the stationary phase
Ion exchange chromatography
152
Application of gel permeation
Separation of triglycerides and fatty acid
153
Separation of compounds is based on their partition between a liquid mobile phase and a liquid stationary phase coated on a solid support
Partition chromatography
154
Example of hydrophobic gel
Sephadex
155
Separation is based on differences between the adsorption and. Desorption of solutes at the surface of a solid particle
Adsorption chromatography
156
Reagents involve
Handling, preparation, storage Proportioning Dispensing
157
Application of partition chromatography
Separation of therapeutic drugs and their metabolites
158
Applications of affinity chromatography
Separation of lipoproteins, carbohydrates, and glycated hemoglobins Separation and preparation of larger quantities if proteins and antibodies for study
159
Factors that serve to drive laboratory automation
``` 1 turnaround time (TAT) demands 2 specimen integrity 3 staff shortages 4 economic factors 5 less maintenance, calibration, downtime 6 faster start-up times 7 24/7 uptime 8 throughput 9 computer and software technology 10 primary tube sampling 11 increasing number of different analytes/methods on a system 12 reducing laboratory errors 13 number of specimens 14 types of fluids 15 safety and environmental concerns ```
160
Types of analytical error
Random Systematic Total Idiosyncratic
161
Refers to assay errors from all sources
Analytical errors
163
Application of ion exchange chromatography
Separation of amino acids and nucleic acids
164
Monitor and maintain required temperatures during incubation
Electronic thermocoupler | Thermistor
165
Not predictable error
Random
166
One direction error
Systematic
167
Random and systematic error
Total
168
Focuses on sample and specimen processing
Pre-analytic stage
168
Advantages of automating laboratory testing
1 increasing the quality of pre-analytic steps 2 reducing error rates 3 reducing operator exposure to potentially hazardous biologic materials 4 eliminating repetitive stress injuries
169
Pre-analytic stage before
Brought to the laboratory by blood drawers
170
Pre-analytic stage at present
Use of pneumatic tubes
172
Nonmethodologic error
Idiosyncratic
174
Tasks in the analytic stage of laboratory testing
``` 1 sample introduction and transport to cuvet/cup 2 addition of reagent 3 mixing of sample and reagent 4 incubation 5 detection 6 calculations 7 readout and result reporting ```
175
Incubation in automated analyzers
Heating air, water, metal
176
Data processing by computers in post-analytic stage
Data acquisition Calculations Monitoring Displaying data
178
Mixing in automated systems include
``` 1 magnetic stirring 2 rotating paddles 3 forceful dispensing 4 use of ultrasonic energy 5 vigorous lateral displacement ```
179
Sources of problems in sample introduction
Formation of clot attached to probe Inadequate or short sample Carry-over
180
Stages of laboratory testing errors
Pre-analytic (61.9%) Analytic (15%) Post-analytic (23.1%)
180
Material of probes used in sample introduction
Thin, stainless steel
181
Computers can
1 perform corrections on data, subtract blank responses, determine first-order linear regression for slope and intercept 2 monitor results against reference values 3 test control data against established QC protocols 4 display patient results, QC data, maintenance and instrumentation operation checks 5 be linked to other computers
181
Examples of instruments used in sample introduction
Peristaltic pumps | Positive-liquid displacement pipets
181
Only manufacturer's reagents
Closed reagent system

Clinical Chemistry Lecture 2 (4 decks)

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