Analytical Method Flashcards
transmitted by electro waves that are characterized by their frequency and wavelength
energy
distance between 2 successive peaks and it is expressed in nanometer (nm)
wavelength
number of vibration of wave motion per second
frequency
- technique used to determine the concentration of colored compounds in solutions
- principle that the concentration of a substance is proportional to the intensity
of the color of the solution
colorimetry
primary analytical utility of spectrophotometry or filter photometry is the isolation of discreet portions of the spectrum for purposes of measurement
photoelectric colorimetry
measurement of light intensity in a narrower wavelength.
spectrophotometric measurement
measurement of light intensity
Photometric measurement
involves the measurement of the light transmitted by a solution to determine the
concentration of the light-absorbing in the solution
Spectrophotometry
- simplest type of absorption spectrometer
- designed to make one measurement at a time at one specified wavelength
- absorption maximum of the analyte must be known in advance when a single instrument is used
Single beam spectrophotometer
- an instrument is this the monochromatic light in the two components - one through the sample, one through a reference solution or blank
- additional beam corrects for variation in light source intensity
- The absorbance of the sample can be recorded via electrical
the output of the sample beam
Double beam spectrophotometer
- an analytical instrument used to quantify the analytes in a given sample using a light beam
- absorption of light by the sample
spectrophotometer
2 photodetectors, for the
sample beam and reference
beam
double beam in space
one photodetector and alternately passes the monochromator through the sample cuvet and the reference cuvette using a
chopped rotating sector mirror
double beam in time
provides the initial light that will pass through the sample
Radiant Energy/Light source
emits radiation that changes in intensity; widely used in the laboratory
Continuum source
most commonly used light source in the lab
Tungsten
used to provide UV radiation in analytic specimens
Deuterium
discharge lamp, produces a continuous source of radiation, which covers
both the invisible and visible range
Xenon lamps
emits limited radiation and wavelength, line source emits discrete lines widely used
in atomic absorption and fluorescent spectroscopy
Line source
- minimizes unwanted or stays light and prevents the entrance of scattered light into the monochromator system
Entrance slit
most common cause of loss of linearity at high analyte concentration
ERROR
it isolates specific or individual wavelengths of light from a broad spectrum produced by the light source
Monochromator
- wedge-shaped pieces of glass, quartz or sodium chloride
- can be rotated, allowing only the desired wavelength to pass through an exit slit.
- A narrow light focused on a prism is refracted as it enters the more dense glass
prisms
- most commonly used; better resolution than prism
- made by cutting grooves (parallel grooves) or slits into an aluminized surface of a flat piece of crown glass
- wavelengths are bent as they pass a sharp corner
Diffraction gratings
- simple, least expensive, not precise but useful
- made by placing semi-transparent silver films on both sides of a dielectric such as magnesium fluoride
- usually pass a wide band of radiant energy and have a low transmittance of the selected
wavelength
filters
optical components used in spectrometers and other optical devices to disperse light into
its component wavelengths
Holographic gratings
- controls the width ofthe light beam (bandpass)
- narrow fraction of the spectrum to reach the sample cuvette
- Spectral purity of the spectrophotometer is reflected by the bandpass, that is, the narrower the bandpass, the greater the resolution.
exit slits
- also called absorption/analytical/sample cell
- holds the solution whose concentration is to be measured
cuvettes
kinds of cuvettes: most commonly used (can be used in 350-2000nm)
Alumina silica glass
used for measurement of solution requiring visible and ultraviolet spectra
Quartz/plastic
detects and converts transmitted light into photoelectric energy. It detects the amount of
light that passes through the sample in the cuvet
Photodetector
kind of detector:
- simplest detector; least expensive; temperature-sensitive
- used in filter photometers with a wide bandpass.
barrier layer cell
kind of detector:
- contains cathode and anode enclosed in a glass case
- photosensitive material that gives off electron when light energy strikes it
- requires an external voltage for operation
Phototube
kind of detector:
- most commonly used detector - measures visible and UV regions
- excellent sensitivity and has a rapid response - detects very low levels of light
Photomultiplier tube (PMT)
kind of detector:
- not as sensitive as PMT but with excellent linearity.
- measures light at a multitude of wavelengths - detects less amount of light.
- lower dynamic range and higher noise compared to PMT
- most useful as a simultaneous multichannel detector.
Photodiode
kind of detector:
- displays output of the detection system
Meter or read-out device
- states that the concentration of the unknown substance is directly proportional to the absorbed
light and inversely proportional to the amount of transmitted light - mathematically establishes the relationship between concentration and absorbance.
- Beer’s Law
- amount of light absorbed
- proportional to the inverse log of
transmittance
absorbance
ratio of the radiant energy transmitted (T) divided by the radiant energy incident on the
sample
Percent Transmittance
- blank contains serum but without the reagent to complete the assay
- blank corrects absorbance caused by the color of the reagents
- measures absorbance of the sample and reagent in the absence of the end of product, and corrects the measurement for optical interference (like hemoglobin) absorbing the wavelength of measurement
Blanking Technique
measures the light emitted by a single atom burned in a flame.
Flame Emission Photometry (FEP)
measures the light absorbed by atoms dissociated by heat.
Atomic Absorption Spectrophotometry (AAS)
The unknown sample is made to react with a known solution in the presence of an indicator.
volumetric
- measuring abundant large particles (proteins) and bacterial suspensions.
- depends on specimen concentration and particle size. The measurement of reduction of light is due to particle formation
TURBIDIMETRY
- measuring the amount of antigen-antibody complexes (proteins)
- It determines the amount of scattered light by a particulate matter suspended in a
turbid solution
nephelometry
- migration of charged particles in an electric field.
- separates proteins on the basis of their electric charge densities.
electrophoresis
a net charge that can be either positive or negative depending on pH conditions
Amphoteric
movement of buffer ions and solvent relative to the fixed
support
Electroendosmosis/Endomosis
migration of small charged ions
lontophoresis
migration of charged macromolecules
Zone electrophoresis
- separates molecules by migration through a pH gradient
- ideal for separating proteins of identical sizes but with different net charges.
Isoelectric Focusing
- sample molecules are separated by electro-osmotic flow (EOF)
- utilizes nanoliter quantities of specimens.
capillary electrophoresis
the involves separation of soluble components in a solution by specific differences in physical chemical characteristics of the different constituent
CHROMATOGRAPHY
- used for fractionation of sugar and amino acid.
- Sorbent (stationary phase) - Whatman paper
paper chromatography
- a semiquantitative drug screening test
- Sample components are identified by comparison with standards on the same plate
Thin layer chromatographty
- used for separation of steroids, barbiturates, blood, alcohol and lipids.
- useful for compounds that are naturally volatile or can be easily converted into a volatile
form
gas chromatography
- based on the fragmentation and ionization of molecules using a suitable source of energy
- can also detect structural information and determination of in molecular weight.
Mass Spectroscopy (MS)
- gold standard for drug testing
- also used for xenobiotics, anabolic steroids, and pesticides.
- uses an electron beam means to split the drug emerging from the column into its component ions -
drugs are detected fragments neans of the presence of decomposition fragmerts which arise after degradation of the analytes.
Gas Chromatography-Mass Spectroscopy (GC-MS)
- based on the distribution of solutes between a liquid mobile phase and a stationary phase
- HPLC is the most widely used liquid chromatography
Liquid Chromatography
- uses pressure for fast separations, controlled temperature, in-line detectors and gradient elution technique
High Performance Liquid Chromatography (HPLC)
- detecting nonvolatile substances in body fluids.
- utilized to confirm positive results from screening of illicit drug
- a complementary method to GC-MS
Liquid Chromatography-Mass Spectroscopy (LC-MS)
separates molecules based on differences in their size and shape
Gel/Gel Permeation/Gel Filtration/Size Exclusion /Molecular Sieve Chromatography
for separation of enzymes, antibodies and proteins - example: dextran and agarose
Hydrophilic gel (Gel Filtration)
separation of triglyceride and fatty acid - example: sephadex
Hydrophobic gel (Gel Permeation)
exchange of sample ions and mobile phase ions with the charge groups of the stationary phase.
- Used to separate amino acids,
proteins, nucleic acids.
Ion exchange chromatography
- separation of compounds are based on their partition between the liquid mobile phase in the
liquid stationary phase coated in solid support - used to separate therapeutic drugs and their metabolites.
Partition chromatography
- uses immobilized biochemical ligands as the stationary phase.
- separation uses the lock-key binding which is present widely in biologic systems.
- used to separate lipoproteins, carbohydrates and glycated hemoglobins; antibodies
Affinity chromatography
- separation is based on the differences between the adsorption and the desorption of solutes at the surface of a solid particle
- compounds are adsorbed to a solid support such a silica or alumina.
Adsorption chromatography
- measures the amount of light intensity present over a zero background
- determines the amount of light emitted by a molecule after excitation by electromagnetic radiation
FLUOROMETRY/MOLECULAR
LUMINESCENCE
- refers to the process by which the intensity of fluorescence is reduced due to various interactions between the fluorescent molecules and other substances or environmental
factors - can affect the accuracy and reliability of fluorescence-based assays, which are commonly used to measure the concentration of analytes in biological sample
Quenching
- a surface phenomenon in which molecules, ions, or particles from a fluid (gas or liquid) adhere to the surface of a solid or a liquid
- process differs from absorption, where the substance permeates or is uniformly distributed throughout the bulk of the solid or liquid
Adsorption
- differs from fluorescence and phosphorescence in that the emission of light is created from a
chemical or electrochemical reaction, and not from absorption of electromagnetic energy - 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
CHEMILUMINESCENCE
- the measurement of the osmolality of an aqueous solution such as serum, plasma, or
urine. - based on measuring changes in the colligative properties of solutions that occur owing to variations in particle concentration
- OSMOMETRY
- measurement of electrical potential due to the activity of free ions - change in voltage
indicates activity of each analyte - measurement of differences in voltage (potential) at a constant current
Potentiometry
- measurement of the amount of electricity (in coulombs) at a fixed potential
- electrochemical titration in which the titrant is electrochemically generated and the
endpoint is detected by amperometry
Coulometry
- measurement of the current flow produced by oxidation-reaction.
Amperometry
- measurement of differences in current at a constant voltage
Polarography
- measurement of current after which a potential is applied to an electrochemical cell
- allows sample to be preconcentrated, thus utilizing minimal analyte
Voltammetry
