PRELIM 1: ANALYTICAL TECHNIQUES AND INSTRUMENTATION Flashcards
is the highest point
Crest or peak
is the lowest
point
trough
is the number of waves that pass an observation
point in a unit of time
Frequency
is the distance of two peaks/crest or troughs
when light travels in a wavelike manner
Wavelength
is the distance between two adjacent peak and
trough
Amplitude
is inversely proportional to the frequency of
the light wave
Wavelength
is inversely proportional to the wavelength of light
Energy
Wavelength or frequency of the electromagnetic waves are perceived as
color or hue
Height or amplitude of the electromagnetic waves are
perceived as
intensity or brightness
There are two kinds of wavelengths: ______which
can be observed at 340-700nm and the_____
which could either be Ultraviolet with a wavelength of less
than 400nm or Infrared with a wavelength of greater than
700 nm.
Visible Spectra, Invisible Spectra
Principle: Measures the amount of light transmitted to determine the concentration of the light-absorbing substance in the solution; the measurement of the light transmitted by a
solution to determine the concentration of the light-absorbing
substance in the solution
Spectrophotometry
states that the concentration of
substance is directly proportional to the amount of light absorbed but inversely proportional to the logarithm of transmitted light
Beer-Lamberts Law
= amount of light
absorbed
Absorbance (OD-optical density)
-provides electromagnetic radiation as visible,
infrared, or UV light
Light source
type of light source can emit UV light
Mercury Vapor Lamp
type of light source ideal for emission
of light within the visible region (iodide prolongs stability of Tungsten); produces energy wavelength from 340-700nm (visible region); used for moderately
diluted solution
Tungsten/Tungsten-iodide lamp
type of light source energy wavelength UV
range (down to 165nm)
Deuterium Discharge Lamp
Types of light source
-Tungsten/Tungsten-iodide lamp
-Mercury Vapor Lamp
-Deuterium Discharge Lamp
-Infrared Energy Source
-Quart Halide Lamp
-Mercury vapor Lamp
-hallow cathode lamp
type of light source above 800 nm
Infrared Energy Source
- reduces stray light and prevents scattered light from entering the monochromator
Entrance Slit
type of light source contains small amt of halogen such as iodine to prevent the decomposition of vaporized tungsten
Quart Halide Lamp
type of light source exists narrow bands of energy at well defined places in the spectrum UV and visible light
Mercury Vapor Lamp
Spectophotometer components
-light source
-entrance slit
-monochromator
-exit slit
-analytical cell
-detector
-meter
type of light source consists of a gas-tight
chamber containing anode, a cylindrical cathode and
insert gas such as helium
Hollow Cathode Lamp
- isolates the specific wavelength of choice
Monochromator
types of monochromator
-prism
-gratings
-colored filters
-interference filters
commonly used type of monochromator
diffraction gratings
-wedge-shaped pieces of glass, quartz, or sodium chloride that allows transmission of light wherein each side of the prism has different thickness allowing selection of wavelength of light
-disperses white light into a continuous spectrum of colors based on variation of refractive index for different wavelength
Prism
-has small grooves cut at such an angle that each grooves behave like a very small prism and the wavelengths are bent as they pass a sharp corner
-separate white light into various color comp.
Gratings
-made of glass that absorbs some portion of the electromagentic spectrum and transmit others wherein
light energy is absorbed by dye components on the class and is dissipated as heat
-band pass is 35-50nm or more
Colored Filters
- used to hold the solution in the instrument
whose concentration is to be measured
Analytical Cell
-enhances desired wavelength by constructive
interference and eliminates others by destructive
interferences
-utlizes the wave cx of light to enhance the intensity of
the desired wavelength by constructive interference and
eliminates others by destructive interference and reflections
-band pass is 10-20nm
Interference Filters
type of analaytical cell for alkaline solution that do not etch
glass
Borosilicate Glass
type of analaytical cell best for wavelength below 320nm
Quartz or Plastic
type of analaytical cell best for visible light
Aluminum Silica Glass
type of analaytical cell best for acidic solution
Soft glass
- converts transmitted light energy into an equivalent
amount of electrical energy
Detector
type of detector composed of film of light sensitive
material; no power source needed
Barrier layer cells
type of detector has photosensitive material that gives off electron when light energy strikes it; requires
an outside voltage for operation
Photoemission tube
type of detector used a series of electrodes to
internally amplify the photosignal before leaving the tube
Photomultiplier tube
Principle: measures the light emitted when electrons in an
atom become excited by heat energy produced by the flame
Flame Emission Spectrophotometry
simplest method of displaying output
of the detection system
Meter (read out device)
Measures electrolytes with a 1+ charge: Na, K, Li
Flame Emission Spectrophotometry
Excited atoms return to ground state by emitting light energy
that is characteristic of that atom
Flame Emission Spectrophotometry
Analyte and color emitted by Flame Emission Spectrophotometry
Sodium filter
Potassium filter
Lithium filter
Potassium filter transmits only
violet light (367 nm)
Lithium filter-transmits only
red light (767nm)
Sodium filter transmits only
yellow light (589 nm)
Components of FES
-Flame
-gases
-automizer or burner
-Interference Filter
-photocell
-breaks the chemical bonds to produce atoms
-source of energy that will be absorbed by the atoms to enter
the excitation state
Flame
-breaks up the solution into finer droplets so that the atom will
absorb heat energy from the flame and get excited
Atomizer or Burner
-using a mixture of hydrogen and oxygen gas (acetylene,
propane or natural gas)
Gases
aspirate sample directly into
flame
Total Consumption Burner
- involves the gravitational feeding of solution
Premix Burner
- serves as monochromator in FES
Interference Filter
- serves as photodetector in FES
Photocell
is the preferred internal standard which also acts as a radiation buffer in FES
Lithium
Principle: measures concentration of the element by
detecting absorption of electromagnetic radiation by atoms
Atomic Absorption Spectrophotometry
The elements are not excited but are dissociated from their
chemical bonds and placed in the unionized, unexcited ground state
Atomic Absorption Spectrophotometry
Measures electrolytes with a 2+ charge: Ca2+, Mg2+
Atomic Absorption Spectrophotometry
Atomic Absorption Spectrophotometry components
-light source
-mechanical rotating chopper
-burner
-monochromator
-detector
-read out device
- modulates light beam
coming from the light source
Mechanical Rotating Chopper
A burner that uses flame to dissociate the chemical bonds and
form free unexcited atoms
AAS
Principle: Unknown samples are made to react with a known
solution in the presence of an indicator
Sample tests: _________
Volumetry/Titrimetry
Schales & Schales method;EDTA Titration
Principle: it is the isolation of the pure form of the sample
and its derivatives and the determination of its dry weight
Sample tests: _______
Gravimetry
Lipid determination
Principle: Measures the amount of light blocked by a
suspension of particular matter as light passes through the
cuvette
Turbidimetry
Principle: Measures the amount of light scattered by small
particles at an angle to the beam incident on the cuvette
Nephelometry
Flow Cytometry interpretation cell granularity and nuclear irregularity=
90° angle scatter
Principle: measures multiple properties of cells suspended
in a moving fluid medium
Flow Cytometry
Flow Cytometry interpretation
cell size=_______
Forward light scatter
a phase in chromatography that carries the complex mixture
mobile phase
the constituents of the mixture are separated by a continuous
redistribution between two phases; mobile phase and stationary phase
Chromatography
Principle: involves the separation of a mixture on the basis
of specific differences of the physical and chemical
characteristics of the different components on a supporting
medium
Chromatography
a phase in chromatography where the mobile phase flows
stationary phase
Principle: A spot of the substance fractioned is placed on the paper just above the solvent level
The organic solvent moves up through the paper by
capillary action and variations in the sample move at
different rates
Paper Chromatography
Types of Chromatorgraphy
-Paper Chromatography
-Thin Layer Chromatography
-Liquid-Liquid Chromatography
-Ion Exchange Chromatography
-Column Chromatography
-Gel Chromatography
-Gas Chromatography
Basis of Separation:
o Rate of diffusion
o Solubility of solute
o Nature of the solvent
Paper Chromatography
Clinical use: fraction of sugars, AA and barbituates
Paper Chromatograph
Same principle as paper chromatography but differs in
the sorbent used
SORBENT: thin plastic plates impregnated to a layer of
silica gel, alumina, polyacrylamide gel or starch gel
Thin Layer Chromatography
Principle: the use of a resin (the stationary solid phase) is used to covalently attach anions or cations onto it
Ion Exchange Chromatography
Principle: separation of substances according to their solubility in an organic/non-polar solvent and in an
aqueous/polar solvent
“Like Dissolves Like”
Liquid-Liquid Chromatography
Clinical use: fractionation of barbituates and lipids
Liquid-Liquid Chromatography
Basis of Separation:
o difference in pH
o polarity of solvent
Column Chromatography
Principle: adsorption of the solutes of a solution through a stationary phase and separates the mixture into individual components
Column Chromatography
Clinical use: Fractionation of sugars
Column Chromatography
Principle: the use of a resin (the stationary solid phase)
is used to covalently attach anions or cations onto it
Gel Chromatography
Principle: separating and measuring nanograms and pictogram amounts of volatile substance
Gas Chromatography
Basis of Separation:
o Molecular weight & size
o Charge of ions
o Hydrophobicity of the molecules
Gel Chromatography
Kinds of GC
_______- sorbent is solid
w/ a large surface
_______-sorbent is a
non-volatile liquid
Gas-Solid Chromatography
Gas-Liquid Chromatography
Basis of Separation:
o sample volatility
o rate of diffusion into liquid layer of the column
packing
o solubility of sample in the liquid layer
Gas Chromatography
Clinical use: drug screening and drug analysis
fractionation of steroids, lipids, barbituates, blood
alcohol and other toxicologic substances
Gas Chromatography
Principle: measurement of difference in current at a
constant voltage
______: relationship between the difference in
current and voltage
Polarography
Ilkovic Equation
Principle: measures the difference in voltage at a constant
current
_______: relationship between the measured voltage and the unknown concentration
Potentiometry
Nerst Equation
Principle: measures the current flow between two nonpolarizable electrodes between a known electrical potential is
established
Conductometry
Principle: measures the amount of current that flows when
constant voltage is applied the measuring electrode
Amperometry
Principle: Measures the fluorescence or the energy
emission that occurs when a certain compound absorbs
electromagnetic radiation, become excited and then return to an energy state that is usually higher than their original level
-Emitted light has longer wavelength than the incident/excited
light due to the loss of energy during collision
Fluorimetry
Principle: measures the amount of electricity (coulombs) at a
fixed potential
_____: number of coulombs consumed can be
directly related to the concentration of the unknown
Coulometry
Faraday’s Law
Fluorimetry Main problem w/ fluorescence:
Quenching
-has 2 monochromators
fluorimetry
Principle: migration or movement of charged particles in an
electric field
Electrophoresis
- strip with a clear
plastic backing w/ a coating of cellulose acetate particles attached to it
Disadv: becomes brittle when dried
Cellulose Acetate Electrophoresis
- earliest support media
Disadv: paper is fragile and easily damaged staining of
protein
Paper Electrophoresis
Disadv: electric neutrality; separation is strictly on the
basis of electric charge and uniformity of material size
Agarose Electrophoresis
- good for large samp
Disadv: fragile and unable to store results permanently
Starch Gel Electrophoresis
- uses protein
size as the major factor in the separation process and
the net charge of proteins
Polyacrylamide Gel Electrophoresis
Electrophoresis Specimens:
Serum, Urine, Cerebrospinal Fluid
Clinical Use: analysis of proteins (serum) that can provide
quick and useful information regarding the presence or
absence of disease entities
Electrophoresis
Basic Approaches in Automation
-CONTINUOUS FLOW ANALYZER
-DISCRETE ANALYZER
-CENTRIFUGAL ANALYZER
-Sequential analysis
-Uniformity in test performance
CONTINUOUS FLOW ANALYZER
-Separate analysis
-Most popular and versatile analyzer
DISCRETE ANALYZER
-Batch analysis
-Centrifugal force moves the reagents and sample to a mixing chamber, into a cuvette, passing a light beam
and measuring the absorbance
CENTRIFUGAL ANALYZER
