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Clinical Chemistry Lecture 2 > Analytical Methods > Flashcards

Analytical Methods Flashcards

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
Q

May either be single-beam or double-beam

A

Spectrophotometer

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26
Q

Instruments used in spectrophotometry

A

Filter photometers

Spectrophotometers

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27
Q

What is a single-beam spectrophotometer?

A

It makes one measurement at a time at one specified wavelength.

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28
Q

What does Beer’s law state?

A

The concentration of a substance is directly proportional to the light absorbed or inversely proportional to the logarithm of the transmitted light.

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29
Q

Light source of a spectrophotometer

A

Incident light

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30
Q

It determines the color of light seen by the eye

A

Wavelength of light

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31
Q

Types of incident light used by a spectrophotometer

A 
1 Continuum
Deuterium (< 300 nm)
Tungsten (400-800 nm)
Xenon
Change in intensity
Adapt

2 Light
Cathode lamp
Does not adapt

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32
Q

Selects wavelength in a spectrophotometer

A

Monochromator

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33
Q

Types of monochromator

A

Colored glass filters
Interference filters
Prism
Diffraction grating

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34
Q

What is a double-beam spectrophotometer?

A

It splits monochromatic light into two components and records absorbance of a sample directly.

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35
Q

Most common material used in making cuvets

A

Silicate

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36
Q

Affect the results when present in the sample holder

A

Scratch

Alkaline

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37
Q

Types of sample holder

A

Square or round
Glass
Quartz

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38
Q

Parts of a spectrophotometer

A 
Light source
Collimator
Monochromator
Slit
Sample holder
Photodetector
Read-out system
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38
Q

Range detected by glass cuvets

A

Visible range

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38
Q

Detected by quartz cuvets

A

UV Radiation

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39
Q

Types of read-out system

A

Moving needle

Digital display of output

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40
Q

Function of a collimator

A

Limits stray light

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43
Q

Chromatography

A

Gas
Liquid
Thin layer

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44
Q

Function of an external slit

A

Limits the band pass

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45
Q

Examples of photodetectors

A

Photocell
Phototube
Photomultiplier
Photodiode

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45
Q

Applications of atomic absorption spectrophotometry

A

Electrolytes (Na, K, Ca, H, Cl)

Dissolved gases

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46
Q

It converts transmitted radiant energy into an equivalent amount of electrical energy

A

Photodetector

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46
Q

Light source of atomic absorption spectrophotometers

A

Hollow cathode lamp

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47
Q

Function of beam choppers of atomic absorption spectrophotometers

A

Used to modulate the light source

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48
Q

It converts ions to atoms in atomic absorption spectrophotometry

A

Chopper

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49
Q

Special application of atomic absorption spectrophotometry

A

Detects small elements when concentration is too low

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52
Q

It excites a molecule at the ground state Eo lebel to a higher excited energy level E1

A

Light absorption

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53
Q

Requirement of mass spectrometry before a compound can be detected and quantified

A

Must be isolated by another method (GC or HPLC)

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53
Q

Applications of Gas Chromatography-Mass Spectrometry

A

Gold standard for drug testing

Proteomics

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54
Q

Classification methods

A

Separation
Qualitative
Quantitative

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55
Q

Major steps in mass spectrometry

A

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

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56
Q

Principle of fluorometry

A

Measures the amount of light emitted by a molecule after excitation by electromagnetic radiation

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58
Q

Electroanalytic methods

A

Electrophoresis
Potentiometry
Amperometry
Voltammetry

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58
Q

Vibrational energy losses

A

Collisions

Heat losses

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59
Q

These molecules can fluoresce

A

Organic molecules with conjugated double bonds

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60
Q

Components of a fluorometer

A

Light source
Monochromator
Detector
Read-out system

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61
Q

Light sources of fluorometers

A

Mercury arc discharge lamp

Xenon arc tube

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62
Q

Forward light scatter nephelometry

A

Rayleigh-Debye type

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63
Q

Examples of monochromators of fluorometers

A

Diffraction grating

Filter

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64
Q

Examples of detectors used in fluorometry

A

Phototube

Photomultiplier tube

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66
Q

Principle of atomic absorption spectrophotometer

A

Measures the absorption of light of a unique wavelength by atoms in the ground state

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67
Q

Types of monochromator used in fluorometry

A

Primary or grating filter

Secondary filter

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68
Q

It allows passage of light of the proper wavelength for absorption of molecule

A

Primary filter or grating

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69
Q

It transmits light of the specific wavelength emitted by the sample

A

Secondary monochromator

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70
Q

Factors that affect fluorescence

A

pH changes
Temperature
Length of time of exposure
Concentration

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71
Q

Clinical application of fluorometry

A

Measurement of porphyrins, magnesium, calcium, and catecholamines

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72
Q

Application of chemiluminescence

A

Immunoassays

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73
Q

Advantages of chemiluminescence

A

Subpicomolar detection limits
Speed
Ease of use
Simple instrumentation

74
Q

Disadvantage of chemiluminescence

A

Impurities can cause background signal that degrades sensitivity and specificity

75
Q

Examples of photodetectors used in chemiluminescence

A

Photomultiplier tube

Luminometer

76
Q

It is taken as the signal in chemiluminescence

A

Integral of the entire peak

77
Q

Principle of chemiluminescence

A

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
Q

Organic compounds in chemiluminescence

A

Luminol
Acridium esters
Dioxetanes by oxidants hydrogen peroxide, hypochlorite or oxygen

79
Q

It involves oxidation of an organic compound characterized by a rapid increase in intensity of emitted light followed by a gradual decay

A

Chemiluminescence

80
Q

Application of nephelometry

A

Measurement of Ag-Ab reactions

81
Q

It comes from the species

A

Chemiluminescence

82
Q

Produced as part of the chemical energy generated and decay to a ground state with the emission of photons

A

Excited intermediates

83
Q

No excitation radiation is required and monochromators are not needed

A

Chemiluminescence

84
Q

Emitted radiation is measured with a photomultiplier tube and the signal is related to the analyte concentration

A

Chemiluminescence

85
Q

Components of a nephelometer

A 
Light source
Collimator
Monochromator
Simple cuvet
Stray light trap
Photodetector
86
Q

Principle of nephelometry

A

Measurement of the light scattered by a particulate solution

87
Q

It detects light that is scattered at various angles and the signal that the scattered light yields is amplified

A

Nephelometry

88
Q

Dependent on wavelength of incident light and particle size

A

Light scattering

89
Q

Wavelength if light > particle diameter

A

Symmetrical

90
Q

Wavelength of light < particle diameter

A

Forward

91
Q

Wavelength of light = particle diameter

A

More forward

92
Q

Diameter of most Ag-Ab complexes

A

250-1500 nm

92
Q

Wavelengths used for most Ag-Ab complexes

A

320-650 nm

93
Q

Sorbent of paper chromatography

A

Whatman paper

94
Q

Principle of turbidimetry

A

Measures reduction in light transmission due to particle formation

95
Q

It detects light transmitted in the forward direction

A

Turbidimetry

96
Q

It is dependent on the specimen concentration and particle size

A

Amount of light absorbed by a suspension of particles

97
Q

Instruments used in turbidimetry to measure solutions for quanitification

A

Visible photometers

Visible spectrophotometers

98
Q

Applications of turbidimetry

A

Protein measurement in CSF and urine
Detection of bacterial growth in broth cultures
Measurement of antibiotic sensitivities
Detection of clot formation

99
Q

Principle of electrophoresis

A

Separation of charged compounds based on their electrical charge

100
Q

Components of electrophoresis instrument

A 
Electrical power
Support medium
Buffer
Sample
Detecting medium
101
Q

Factors affecting rate of migration in electrophoresis

A 
Net electric charge of the molecule
Size and shape of the molecule
Electric field strength
Nature of the supporting medium
Temperature of operation
102
Q

Produced by the flow of ions when a voltage is applied to a salt solution

A

Electrical current

103
Q

Supporting media used in electrophoresis

A 
Paper electrophoresis
Starch gel
Cellulose acetate*
Agarose gel*
Polyacrylamide gel*
104
Q

Separates by surface charge and molecular size

A

Starch gel

105
Q

Separates by molecular size

A

Cellulose acetate

106
Q

Separates by electrical charge and does not bind protein

A

Agarose gel

107
Q

Neutral supporting media

A

Agarose gel

Polyacrylamide gel

108
Q

Used to study isoenzymes

A

Polyacrylamide gel

109
Q

Neutral; separates on the basis of charge and molecular size

A

Polyacrylamide gel

110
Q

Stains for visualization of fractions in electrophoresis

A 
Amido black*
Ponceau S*
Oil red O
Sudan black*
Fat red 7B*
Coomassie blue
Gold/silver stain
111
Q

It measures the absorbance of the stain on a support medium

A

Densitometer

112
Q

Components of a densitometer

A

Light source
Monochromator
Optical system
Photodetector

113
Q

Principle of densitometry

A

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
Q

It is a modification of electrophoresis

A

Isoelectric focusing

114
Q

Principle of isoelectric focusing

A

Charged proteins migrate through a support medium that has a continuous pH gradient

115
Q

Applications of isoelectric focusing

A

Detects oligoclonal immunoglobulin bands in CSF

Detects isoenzymes of CK, ACP, ALP in serum

116
Q

It follows the Nernst equation

A

Potentiometry

117
Q

Principle of potentiometry

A

Concentrations of ions in a solution can be calculated from the measured potential difference between 2 electrodes (reference and indicator electrode)

118
Q

What does potentiometry measure?

A

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
Q

What is amperometry?

A

Measurement of the current flow produced by an oxidation-reduction reaction

120
Q

Applications of amperometry

A

Determination of pO2, chloride, and peroxidase

121
Q

Advantages of voltammetry

A

Sensitivity and capability for multi-element measurements (most important)
Consumes minimal analyte

122
Q

Method in which a potential is applied to an electrochemical cell and the resulting current is measured

A

Voltammetry

123
Q

Measures heavy metals like lead

A

Anodic stripping voltammetry

124
Q

Application of coulometry

A

Measurement of chloride ion in serum, plasma, CSF, and sweat samples

125
Q

Faraday’s law

A

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
Q

What is chromatography?

A

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
Q

2 forms of chromatography

A

Planar

Column

129
Q

Examples of planar chromatography

A

Paper

Thin layer

130
Q

Examples of column chromatography

A

Gas

Liquid

131
Q

Sorbent of thin layer chromatography

A

Thin plastic plates impregnated with a thin layer of silica gel or alumina

132
Q

Application of thin layer chromatography

A

Drug screening

133
Q

Advantage of isoelectric focusing

A

It can resolve mixtures of proteins

134
Q

Application of paper chromatography

A

Fraction of sugar and amino acid

135
Q

It measures the quantity of electricity (in coulombs) needed to convert an analyte to a different oxidation state

A

Coulometry

136
Q

Application of gas chromatography

A

Separation of steroids, barbiturates, blood, alcohol, lipids

137
Q

Useful for compounds that are naturally volatile or can be easily converted into a volatile form

A

Gas chromatography

138
Q

Types of stationary phases in gas chromatography

A

Gas-solid

Gas-liquid

139
Q

Separation occurs by differences in absorption at the solid phase surfaces

A

Gas-solid chromatography

140
Q

Separation occurs by differences in solute partitioning between gaseous mobile phase and liquid stationary phase

A

Gas-liquid cheomatography

141
Q

Most widely used liquid chromatography

A

HPLC

142
Q

Based on the distribution of solutes between a liquid mobile phase and a stationary phase

A

Liquid chromatography

143
Q

Advantages of liquid chromatography over gas chromatography

A

1 No need for chemical derivatization of organic compounds
2 Use of lower temperature for separation
3 Easy recovery of a sample

144
Q

Bases of separation in chromatography

A 
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
Q

Separation mechanisms in liquid chromatography

A 
Gel or molecular sieve
Ion exchange
Partition
Affinity
Adsorption
146
Q

Separates molecules based on differences in size and shape

A

Gel or molecular sieve chromatography

147
Q

Uses immobilized biochemical ligands as the stationary phase to separate a few solutes from other unretained solutes

A

Affinity chromatography

148
Q

Other term for hydrophilic gel

A

Gel filtration

149
Q

Application of gel filtration

A

For separation of enzymes, antibodies, proteins

150
Q

Examples of hydrophilic gel

A

Dextran and agarose

151
Q

Exchange of sample ions and mobile-phase ions with the charged group of the stationary phase

A

Ion exchange chromatography

152
Q

Application of gel permeation

A

Separation of triglycerides and fatty acid

153
Q

Separation of compounds is based on their partition between a liquid mobile phase and a liquid stationary phase coated on a solid support

A

Partition chromatography

154
Q

Example of hydrophobic gel

A

Sephadex

155
Q

Separation is based on differences between the adsorption and. Desorption of solutes at the surface of a solid particle

A

Adsorption chromatography

156
Q

Reagents involve

A

Handling, preparation, storage
Proportioning
Dispensing

157
Q

Application of partition chromatography

A

Separation of therapeutic drugs and their metabolites

158
Q

Applications of affinity chromatography

A

Separation of lipoproteins, carbohydrates, and glycated hemoglobins
Separation and preparation of larger quantities if proteins and antibodies for study

159
Q

Factors that serve to drive laboratory automation

A 
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
Q

Types of analytical error

A

Random
Systematic
Total
Idiosyncratic

161
Q

Refers to assay errors from all sources

A

Analytical errors

163
Q

Application of ion exchange chromatography

A

Separation of amino acids and nucleic acids

164
Q

Monitor and maintain required temperatures during incubation

A

Electronic thermocoupler

Thermistor

165
Q

Not predictable error

A

Random

166
Q

One direction error

A

Systematic

167
Q

Random and systematic error

A

Total

168
Q

Focuses on sample and specimen processing

A

Pre-analytic stage

168
Q

Advantages of automating laboratory testing

A

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
Q

Pre-analytic stage before

A

Brought to the laboratory by blood drawers

170
Q

Pre-analytic stage at present

A

Use of pneumatic tubes

172
Q

Nonmethodologic error

A

Idiosyncratic

174
Q

Tasks in the analytic stage of laboratory testing

A 
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
Q

Incubation in automated analyzers

A

Heating air, water, metal

176
Q

Data processing by computers in post-analytic stage

A

Data acquisition
Calculations
Monitoring
Displaying data

178
Q

Mixing in automated systems include

A 
1 magnetic stirring
2 rotating paddles
3 forceful dispensing
4 use of ultrasonic energy
5 vigorous lateral displacement
179
Q

Sources of problems in sample introduction

A

Formation of clot attached to probe
Inadequate or short sample
Carry-over

180
Q

Stages of laboratory testing errors

A

Pre-analytic (61.9%)
Analytic (15%)
Post-analytic (23.1%)

180
Q

Material of probes used in sample introduction

A

Thin, stainless steel

181
Q

Computers can

A

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
Q

Examples of instruments used in sample introduction

A

Peristaltic pumps

Positive-liquid displacement pipets

181
Q

Only manufacturer’s reagents

A

Closed reagent system

Clinical Chemistry Lecture 2 (4 decks)

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