Analytical Methods Flashcards
When is light emitted in fluorometry?
When the molecule has returned to the more stable ground state
Principle of mass spectrometry
Based on fragmentation and ionization of molecules using a suitable source of energy
4 basic disciplines of analytic methods
1 Spectrometry
2 Luminescence
3 Electroanalytic methods
4 Chromatography
Spectrometry
Spectrophotometry
Atomic absorption
Mass spectrometry
Type of optical methods
Absorption
Emission
Polarization
Scattering
Examples of emission methods
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
Luminescence
Fluorescence
Chemiluminescence
Nephelometry
Examples of polarization methods
Fluorescence polarization spectroscopy
Polarimetry
Examples of scattering methods
Nephelometry
Turbidimetry
It describes the radiant energy with wavelengths visible to the human eye
Light
Short wavelength
Gamma rays
X-rays
Longer wavelength
Radio
Microwave
400 nm wavelength
Violet
700 nm wavelength
Red
Human eye
380-750 nm
Measures shorter (uv) or longer (infrared) wavelength
Photometric apparatus
Short wavelength, high frequency
High gamma rays
T or F. When light is absorbed, it is transmitted.
F. When light is not absorbed, it is transmitted.
Used to select the incident wavelength
Filters (photometers)
Prisms or gratings (spectrometers)
UV at 200-380 nm
Near UV
Examples of absorption methods
Atomic absorption Densitometry Fourier transform infrared spectroscopy Photometry Spectrophotometry Reflectance photometry X-ray spectroscopy
UV at < 220 nm
Far UV
Silica used to make cuvets transmits light effectively at wavelengths _______
> /= 220 nm
Principle of spectrophotometry
Measurement of the light transmitted by a solution to determine concentration of light-absorbing substances in solution
May either be single-beam or double-beam
Spectrophotometer
Instruments used in spectrophotometry
Filter photometers
Spectrophotometers
What is a single-beam spectrophotometer?
It makes one measurement at a time at one specified wavelength.
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.
Light source of a spectrophotometer
Incident light
It determines the color of light seen by the eye
Wavelength of light
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
Selects wavelength in a spectrophotometer
Monochromator
Types of monochromator
Colored glass filters
Interference filters
Prism
Diffraction grating
What is a double-beam spectrophotometer?
It splits monochromatic light into two components and records absorbance of a sample directly.
Most common material used in making cuvets
Silicate
Affect the results when present in the sample holder
Scratch
Alkaline
Types of sample holder
Square or round
Glass
Quartz
Parts of a spectrophotometer
Light source Collimator Monochromator Slit Sample holder Photodetector Read-out system
Range detected by glass cuvets
Visible range
Detected by quartz cuvets
UV Radiation
Types of read-out system
Moving needle
Digital display of output
Function of a collimator
Limits stray light
Chromatography
Gas
Liquid
Thin layer
Function of an external slit
Limits the band pass
Examples of photodetectors
Photocell
Phototube
Photomultiplier
Photodiode
Applications of atomic absorption spectrophotometry
Electrolytes (Na, K, Ca, H, Cl)
Dissolved gases
It converts transmitted radiant energy into an equivalent amount of electrical energy
Photodetector
Light source of atomic absorption spectrophotometers
Hollow cathode lamp
Function of beam choppers of atomic absorption spectrophotometers
Used to modulate the light source
It converts ions to atoms in atomic absorption spectrophotometry
Chopper
Special application of atomic absorption spectrophotometry
Detects small elements when concentration is too low
It excites a molecule at the ground state Eo lebel to a higher excited energy level E1
Light absorption
Requirement of mass spectrometry before a compound can be detected and quantified
Must be isolated by another method (GC or HPLC)
Applications of Gas Chromatography-Mass Spectrometry
Gold standard for drug testing
Proteomics
Classification methods
Separation
Qualitative
Quantitative
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
Principle of fluorometry
Measures the amount of light emitted by a molecule after excitation by electromagnetic radiation
Electroanalytic methods
Electrophoresis
Potentiometry
Amperometry
Voltammetry
Vibrational energy losses
Collisions
Heat losses
These molecules can fluoresce
Organic molecules with conjugated double bonds
Components of a fluorometer
Light source
Monochromator
Detector
Read-out system
Light sources of fluorometers
Mercury arc discharge lamp
Xenon arc tube
Forward light scatter nephelometry
Rayleigh-Debye type
Examples of monochromators of fluorometers
Diffraction grating
Filter
Examples of detectors used in fluorometry
Phototube
Photomultiplier tube
Principle of atomic absorption spectrophotometer
Measures the absorption of light of a unique wavelength by atoms in the ground state
Types of monochromator used in fluorometry
Primary or grating filter
Secondary filter
It allows passage of light of the proper wavelength for absorption of molecule
Primary filter or grating
It transmits light of the specific wavelength emitted by the sample
Secondary monochromator
Factors that affect fluorescence
pH changes
Temperature
Length of time of exposure
Concentration
Clinical application of fluorometry
Measurement of porphyrins, magnesium, calcium, and catecholamines
Application of chemiluminescence
Immunoassays
Advantages of chemiluminescence
Subpicomolar detection limits
Speed
Ease of use
Simple instrumentation
Disadvantage of chemiluminescence
Impurities can cause background signal that degrades sensitivity and specificity
Examples of photodetectors used in chemiluminescence
Photomultiplier tube
Luminometer
It is taken as the signal in chemiluminescence
Integral of the entire peak
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
Organic compounds in chemiluminescence
Luminol
Acridium esters
Dioxetanes by oxidants hydrogen peroxide, hypochlorite or oxygen
It involves oxidation of an organic compound characterized by a rapid increase in intensity of emitted light followed by a gradual decay
Chemiluminescence
Application of nephelometry
Measurement of Ag-Ab reactions
It comes from the species
Chemiluminescence
Produced as part of the chemical energy generated and decay to a ground state with the emission of photons
Excited intermediates
No excitation radiation is required and monochromators are not needed
Chemiluminescence
Emitted radiation is measured with a photomultiplier tube and the signal is related to the analyte concentration
Chemiluminescence
Components of a nephelometer
Light source Collimator Monochromator Simple cuvet Stray light trap Photodetector
Principle of nephelometry
Measurement of the light scattered by a particulate solution
It detects light that is scattered at various angles and the signal that the scattered light yields is amplified
Nephelometry
Dependent on wavelength of incident light and particle size
Light scattering
Wavelength if light > particle diameter
Symmetrical
Wavelength of light < particle diameter
Forward
Wavelength of light = particle diameter
More forward
Diameter of most Ag-Ab complexes
250-1500 nm
Wavelengths used for most Ag-Ab complexes
320-650 nm
Sorbent of paper chromatography
Whatman paper
Principle of turbidimetry
Measures reduction in light transmission due to particle formation
It detects light transmitted in the forward direction
Turbidimetry
It is dependent on the specimen concentration and particle size
Amount of light absorbed by a suspension of particles
Instruments used in turbidimetry to measure solutions for quanitification
Visible photometers
Visible spectrophotometers
Applications of turbidimetry
Protein measurement in CSF and urine
Detection of bacterial growth in broth cultures
Measurement of antibiotic sensitivities
Detection of clot formation
Principle of electrophoresis
Separation of charged compounds based on their electrical charge
Components of electrophoresis instrument
Electrical power Support medium Buffer Sample Detecting medium
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
Produced by the flow of ions when a voltage is applied to a salt solution
Electrical current
Supporting media used in electrophoresis
Paper electrophoresis Starch gel Cellulose acetate* Agarose gel* Polyacrylamide gel*
Separates by surface charge and molecular size
Starch gel
Separates by molecular size
Cellulose acetate
Separates by electrical charge and does not bind protein
Agarose gel
Neutral supporting media
Agarose gel
Polyacrylamide gel
Used to study isoenzymes
Polyacrylamide gel
Neutral; separates on the basis of charge and molecular size
Polyacrylamide gel
Stains for visualization of fractions in electrophoresis
Amido black* Ponceau S* Oil red O Sudan black* Fat red 7B* Coomassie blue Gold/silver stain
It measures the absorbance of the stain on a support medium
Densitometer
Components of a densitometer
Light source
Monochromator
Optical system
Photodetector
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
It is a modification of electrophoresis
Isoelectric focusing
Principle of isoelectric focusing
Charged proteins migrate through a support medium that has a continuous pH gradient
Applications of isoelectric focusing
Detects oligoclonal immunoglobulin bands in CSF
Detects isoenzymes of CK, ACP, ALP in serum
It follows the Nernst equation
Potentiometry
Principle of potentiometry
Concentrations of ions in a solution can be calculated from the measured potential difference between 2 electrodes (reference and indicator electrode)
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
What is amperometry?
Measurement of the current flow produced by an oxidation-reduction reaction
Applications of amperometry
Determination of pO2, chloride, and peroxidase
Advantages of voltammetry
Sensitivity and capability for multi-element measurements (most important)
Consumes minimal analyte
Method in which a potential is applied to an electrochemical cell and the resulting current is measured
Voltammetry
Measures heavy metals like lead
Anodic stripping voltammetry
Application of coulometry
Measurement of chloride ion in serum, plasma, CSF, and sweat samples
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)
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
2 forms of chromatography
Planar
Column
Examples of planar chromatography
Paper
Thin layer
Examples of column chromatography
Gas
Liquid
Sorbent of thin layer chromatography
Thin plastic plates impregnated with a thin layer of silica gel or alumina
Application of thin layer chromatography
Drug screening
Advantage of isoelectric focusing
It can resolve mixtures of proteins
Application of paper chromatography
Fraction of sugar and amino acid
It measures the quantity of electricity (in coulombs) needed to convert an analyte to a different oxidation state
Coulometry
Application of gas chromatography
Separation of steroids, barbiturates, blood, alcohol, lipids
Useful for compounds that are naturally volatile or can be easily converted into a volatile form
Gas chromatography
Types of stationary phases in gas chromatography
Gas-solid
Gas-liquid
Separation occurs by differences in absorption at the solid phase surfaces
Gas-solid chromatography
Separation occurs by differences in solute partitioning between gaseous mobile phase and liquid stationary phase
Gas-liquid cheomatography
Most widely used liquid chromatography
HPLC
Based on the distribution of solutes between a liquid mobile phase and a stationary phase
Liquid chromatography
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
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
Separation mechanisms in liquid chromatography
Gel or molecular sieve Ion exchange Partition Affinity Adsorption
Separates molecules based on differences in size and shape
Gel or molecular sieve chromatography
Uses immobilized biochemical ligands as the stationary phase to separate a few solutes from other unretained solutes
Affinity chromatography
Other term for hydrophilic gel
Gel filtration
Application of gel filtration
For separation of enzymes, antibodies, proteins
Examples of hydrophilic gel
Dextran and agarose
Exchange of sample ions and mobile-phase ions with the charged group of the stationary phase
Ion exchange chromatography
Application of gel permeation
Separation of triglycerides and fatty acid
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
Example of hydrophobic gel
Sephadex
Separation is based on differences between the adsorption and. Desorption of solutes at the surface of a solid particle
Adsorption chromatography
Reagents involve
Handling, preparation, storage
Proportioning
Dispensing
Application of partition chromatography
Separation of therapeutic drugs and their metabolites
Applications of affinity chromatography
Separation of lipoproteins, carbohydrates, and glycated hemoglobins
Separation and preparation of larger quantities if proteins and antibodies for study
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
Types of analytical error
Random
Systematic
Total
Idiosyncratic
Refers to assay errors from all sources
Analytical errors
Application of ion exchange chromatography
Separation of amino acids and nucleic acids
Monitor and maintain required temperatures during incubation
Electronic thermocoupler
Thermistor
Not predictable error
Random
One direction error
Systematic
Random and systematic error
Total
Focuses on sample and specimen processing
Pre-analytic stage
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
Pre-analytic stage before
Brought to the laboratory by blood drawers
Pre-analytic stage at present
Use of pneumatic tubes
Nonmethodologic error
Idiosyncratic
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
Incubation in automated analyzers
Heating air, water, metal
Data processing by computers in post-analytic stage
Data acquisition
Calculations
Monitoring
Displaying data
Mixing in automated systems include
1 magnetic stirring 2 rotating paddles 3 forceful dispensing 4 use of ultrasonic energy 5 vigorous lateral displacement
Sources of problems in sample introduction
Formation of clot attached to probe
Inadequate or short sample
Carry-over
Stages of laboratory testing errors
Pre-analytic (61.9%)
Analytic (15%)
Post-analytic (23.1%)
Material of probes used in sample introduction
Thin, stainless steel
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
Examples of instruments used in sample introduction
Peristaltic pumps
Positive-liquid displacement pipets
Only manufacturer’s reagents
Closed reagent system
