GRAVIMETRIC ANALYSIS
This involves the separation of the substance of interest from other components in the matrix and the weighing of the pure substance or its derivatives to obtain the amount present in the original sample. One application is in the determination of total lipids in serum and feces.
Objection to this method:
1. Requires a large sample.
2. Difficulty in separating the substance
3. Time consuming
VOLUMETRIC ANALYSIS
This involves the determination of a substance by reacting it with a measured volume of known standardized solution. This process is commonly called titration.
The principle of volumetric analysis is illustrated with the formula:
V1 x C1 = V2 x C2
Where:
V1 – volume of titrant (ml) required to reach the end point
C1 – known concentration of titrant expressed in equivalents/L or
mEq/L
V2 – volume of unknown (ml) to be measured
C2 – calculated concentration of unknown in equivalents/L or mEq/L
An application of this method of analysis is the Schales and Schales Chloride titration. In performing a titration, a measured volume of standard solution is made to react with a substance whose concentration is to be measured. The chemical reaction then proceeds in a stoichiometric or controlled manner in the presence of an indicator. This indicator has a property of producing a physical change in the solution when the substance determined has reacted quantitatively.
The physical change maybe in the form of:
a. Change in color
b. Change in electrical conduction of the solution
c. Changes in the electrical potential of the solution
d. Changes in the absorbance of indicator
When titrating strong acids or weak acids with strong base, use an indicator that changes color at an alkaline pH (e.g. phenolpthalein). When titrating strong base with strong acids, use indicator that changes color at an acid pH (e.g. methyl orange).
ELECTROMAGNETIC ENERGY
These are radiant energy from short wavelength gamma rays to long wavelength radiowaves. They are photons (discrete energy packets) of energy travelling in a wavelike manner.
· The shorter the wavelength, the higher the electromagnetic energy.
Types of Electromagnetic energies:
a. Cosmic rays
b. Gamma rays
c. X–rays
d. Visible rays
e. Ultra–Violet (UV)
f. Infrared
g. Radio, TV, Microwaves
Wavelength – distance between peaks as light is envisioned to travel in a wavelike manner and is expressed in nanometer (10–9 m).
Units: a. S.I.: nanometer; 1 nm = 10 A; 1 nm = 1 mu
b. C.U.: Angstrom (A), millimicrons (mu)
visible region – wavelength between 400 – 800 nm
infrared region – above 1000 nm
amplitude – distance between peak and trough
· The higher the amplitude, the more intense the light and the more light energy produced at that wavelength.
Color produced by different wavelength at different regions
Wavelength Region name Color observed
<380 UV not visible
380 – 440 visible violet
440 – 500 visible blue
500 – 580 visible green
580 – 600 visible yellow
600 – 620 visible orange
620 – 750 visible red
750 – 2000 short infrared not visible
Analytical methods which utilizes Electromagnetic energy:
a. Colorimetry
b. Emission Flame Photometry
c. Atomic Absorption Spectrophotometry
d. Fluorometry
e. Turbidimetry
f. Nephelometry
g. Scintillation counter
A.COLORIMETRY
Colorimetry is a method of chemical analysis based on the comparisons of colors produced by an unknown and a standard solution when reacted with similar chemical reagents.
Two primary consideration in colorimetric analysis:
1. Quantity of color
2. Intensity of color
This law forms the mathematical basis of colorimetry. It states that the concentration of a substance is directly proportional to the amount of light absorbed or inversely proportional to the logarithm of the transmitted light.
It may also be stated that the absorbance (A) or the optical density (O.D.) of any colored solution is equal to the products of the concentration of the color producing substance (C) times the depth of the solution through which the light travels (L) times a constant (K). The mathematical expression of Beer’s Law is as follows:
A = C x L x K
% Transmittance – ratio of the radiant energy transmitted, divided by the radiant energy incident on the sample.
Forms of colorimetry:
1. Visual colorimetry – involves the comparison of an unknown solution with a series of colored standard solutions using the naked eye. The unknown is diluted until its color matches the standard.
Disadvantages of visual colorimetry:
a. The intensity matching is open to subjective error so that greatest accuracy is not probable.
b. No method of selecting light of the proper wavelength is included, this results in the colored solution acting as its own filter. To compensate for the inaccuracies inherent in visual colorimetry, use standards that are reasonably close to the concentration of the unknown. This simply means that a series of standards should be included with the samples and the standards nearest in color intensity should be chosen.
2. Photoelectric colorimetry – measurement is done by an instrument which measures light intensity by converting light energy to electric energy.
Principle:
Selected light passing through a solution to a greater or lesser extent strikes a photocell or phototube which generates current registered by a galvanometer.
Two general types of photoelectric colorimeter:
a. Filter photometer – so called because the selection of wavelength of color is accomplished by placing a colored glass filter in the path of the light beam. This is also called as abridged spectrophotometer.
b. Spectrophotometer – in this instrument, the light beam strikes a especially constructed diffraction gratings or quartz which breaks the white light up into the spectrum or systematic array of its component color or wavelength. The formed spectrum may be passed along a narrow slit so that only one particular wavelength is allowed to pass through the opening by moving the light source or mirror. This arrangement in the spectrophotometer is called “monochromator” (selection of one color of light).
Two types of spectrophotometer:
a. Single–beam spectrophotometer
b. Double–beam spectrophotometer
(1) Double–beam–in–space
(2) Double–beam–in–time
The essential differences between the terms filter photometer and spectrophotometer is the method by which the selected light is obtained.
Important terms is photoelectric colorimetry:
1. Transmission – is the mathematical comparison of the intensity of color that emerges from the solution with the same light beam as it enters the solution.
2. Transmittance – a comparison of the transmission of colored solution with that of the blank solution. It is a measure of the amount of light allowed to pass through the solution. The values of absorbance (A) and transmittance (T) are reciprocally related.
A = –log (% T/100)
General rules and precautions in photoelectric colorimetry:
1. A blank should be used for each set of determinations. The purpose of the blank is to read out any absorbance due to the reagents. The blank should be prepared in the same fashion and with the same reagents as the specimens to be analyzed.
2. Colorimeter cuvets should be clean and dry when used. This is accomplished by rinsing with volatile solvents like ether or acetone after washing.
3. Cuvets should be free from scratches.
4. The solutions in each tube to be measured should be free from air bubbles. The bubbles can be removed by tapping the tube or a stirring rod may be used if necessary.
5. Close attention should be paid to specific details of operating each instrument.
6. Stray light from windows or overhead light should be avoided as they can cause marked errors in reading.
Parts of a photoelectric colorimeter:
1. Power source – to furnish approximately regulated electrical energy for the operation of the instrument.
a. Battery
b. Transformer
c. Electric Power Supply
2. Light source – provides radiant energy in the form of visible or non–visible light that may pass through the monochromator. The light of proper wavelength is made incident on the analytical cell.
Types of lamps:
a. Tungsten Iodide Lamp – produces energy wavelength from 340–700 nm (visible region) and used for moderately diluted solution.
b. Quartz Hallide Lamp – contains small amount of halogen such as iodine or bromine to prevent the decomposition of the vaporized tungsten from the very hot filament.
c. Deuterium Discharge Lamp – provide energy source with high output in the UV range (down to 165 nm).
Example:
Nerst glower – an electrically heated rod of rare earth elements oxides.
Globar – uses silicon carbide.
e. Mercury Vapor Lamp – emits narrow bands of energy at well defined places in the spectrum (UV and visible).
f. Hollow Cathode Lamp – consists of a gas–tight chamber containing anode, a cylindrical cathode and inert gas such as helium or argon and is commonly used in AAS.
g. Light Amplification by Stimulated Emission of Radiation (LASER) – one example is the phosphorescence.
Types of LASERS and their wavelengths:
(3) Carbon dioxide – 337 nm
(4) Gallium–arsenic diode – 325 nm
(6) Helium–Cadmium – 800–900 nm
(7) Helium–Neon – 633 nm
(8) Organic dye – 400 – 800 nm
(9) Neodymium–Yttrium–Aluminum garnet – 1060 nm
3. Monochromator – a system for isolating radiant energy of a desired wavelength and excluding that of other wavelength.
Spectral bandwidth – width (in nanometer) of the spectral transmittance curve at a point equal to one half the peak transmittance. It also describes the spectral purity of filter or monochromator.
Monochromatic light – light radiation of a single wavelength.
Types of monochromator:
a. Prisms – a wedge–shaped piece of glass or quartz which separates white light into a continuous spectrum by refraction, that is, shorter wavelengths are bent or refracted, more than longer wavelengths.
b. Gratings – made of aluminum–copper alloy on the surface of a flat glass plate, then ruling many small parallel grooves into the metal coating, each groove behaving like small prisms.
(1) Transmission gratings – utilizes a photoresistive surface etched with multiple lines through laser. This etched material is covered with reflective layer to be ready for use. This grating is extremely accurate, have low light scatter and widely used.
(2) Reflection gratings – the lines are engraved on the surface of a mirror, which may be either a polished metal slab or a glass plate on which a thin, metallic film has been deposited.
(3) Echelette – a grating ruled at a specific angle so that a maximum fraction of the radiant energy is directed into wavelengths diffracted at a selected angle. It either gives a blaze at a particular angle or blazed at certain wavelength.
Advantages of gratings over filters:
(1) Produce linear spectrum and therefore maintaining a constant band pass.
(2) Can be used in the regions of spectrum where light energy is absorbed by glass prisms.
c. Glass filters
(2) Narrow–bandpass filters – constructed either with combining two or more sharp–cutoff filters or regular filters or with the use of dielectric material of controlled thickness sandwhich between two thinly silvered pieces of glass.
(4) Neutral tints or gray filters – have constant absorption over a wide spectral range; they have been used as absorbance to replace standard solution.
(5) Colored filters – made of glass that absorbs some portion of electromagnetic spectrum and transmit others. Light energy is absorbed by dye compounds on the glass and is dissipated as heat. It has a band pass of 35–50 nm or more.
(6) Interference filters – utilizes the wavelength character of light to enhance the intensity of the desired wavelength by constructive interference and eliminates other by destructive interference and reflections. It has a bandpass of 10–20 nm.
4. Fiber optics or light pipes – bundles of thin transparent fibers of glass, quartz or plastic that are enclosed within material of a lower index of refraction and that transmit light throughout their lengths by internal reflections.
Advantages:
a. Better directional control of the beam of light within the instrument; this allows for the design and manufacture of miniature and inexpensive optical subsystem for use in automated instruments.
Disadvantages:
a. Greater amounts of stray light.
b. Refractive index changes within glass, quartz or plastic rods.
c. Loss of transmitted energy after continued use in the UV region of the spectrum. This loss of energy is known as solarization and results in a decrease in the optical sensitivity of an instrument.
5. Analytical cell or cuvets – used to hold the solution in the instrument.
Types of cuvet:
a. Borosilicate glass – suitable for measurement in the visible region.
b. Quartz or silica – suitable for measurements below 340 nm.
c. Plastic – designed for disposable, single use application.
Precaution in using cuvets:
a. It should be clean and optically clear. It should be cleaned with detergent or soaked in a mixture of concentrated HCl : water : ethanol (1:3:4). It should never be soaked in dichromate cleaning solution since the solution tends to adsorb onto and discolor the glass.
b. Etches and deposits on the surface affect absorbance values
c. Cuvets used in the visible range are cleaned by copious rinsing with tap water and distilled water while those used in UV range should be free from scratches, fingerprints or residual traces of previously measured substances.
d. Alkaline solutions on prolonged standing dissolves glass and produces etching.
6. Detectors – electron tubes capable of amplifying current by converting transmitted energy into an equivalent amount of electrical energy.
Types of detectors:
a. Barrier–Layer cell or Photocell or Photovoltaic cell – composed of a film of light sensitive material like selenium on iron plate with transparent layer of silver. It requires no external voltage source.
b. Photoemissive tube or Phototube – possess a photosensitive material that gives off electron when light energy strikes it. It is consists of two electrodes (cathode and anode) sealed in an evacuated glass to prevent scattering of photoelectrons by collision with gas molecules. It requires an outside voltage for operation.
c. Photomultiplier tube – has series of electrodes to internally amplify the photosignal before leaving the tube.
7. Readout devices or meter – where electrical energy is displayed.
Types of meters:
a. Direct reading systems – the output of the photocell is used to drive a sensitive meter directly with no further amplification.
b. Null point system – the output of the detector is balanced against the output of a reference circuit.
c. Digital readouts – provide a visual numerical display of absorbances or converted values of concentrations.
B.EMISSION FLAME PHOTOMETRY (EFP)
This analysis is based on the fact that when a solution containing a metal ion is sprayed into a colorless flame, the atoms are raised to a higher energy level and as they return to their ground state, they release energy in the form of light which is characteristics of the element in solution.
Flame photometry is used in clinical chemistry for the determination of electrolytes.
The following electrolytes produces the following colors:
1. Sodium – yellow
2. Potassium – violet
3. Lithium – red
4. Magnesium – blue
5. Calcium – red
Components of EFP:
1. Aspirator – draws sample into flame.
2. Atomizer – breaks up the solution into finer droplets so that the atom will absorb heat energy from the flame and get excited.
Settling agent – minimizes changes in atomizer flow rate due to differences in viscosity of the sample. Viscosity effect is reduced by a dilution of 1:100 or 1:200.
3. Flame – provides energy for excitation.
Flame temperatures for various gas mixtures:
a. Natural gas–air – 1840oC
b. Propane–air – 1925oC
c. Hydrogen–air – 2115oC
d. Acetylene–air – 2250oC
e. Hydrogen–oxygen – 2700oC
f. Natural gas–oxygen – 2800oC
g. Propane–oxygen – 2850oC
h. Acetylene–oxygen – 3110oC
Argon inductively coupled plasma (ICP) torch – commercially available excitation source for EFP which is coupled to a radiofrequency generation that serves as the means to excite ions and molecules to energy states that will produce light emission.
Other purpose of flame in EFP:
a. Breaks the chemical bond to produce atoms.
b. Source of energy absorbed by the atoms to enter an excited state.
4. Interference filters as monochromator
a. Sodium filter – 589 nm
b. Potassium filter – 767 nm
c. Lithium filter – 761 nm
5. Detector – photocell is used.
Types of EFP:
1. Direct EFP – calibrated solutions of sodium or potassium were atomized or aspirated directly into the flame to provide a series of meter readings against which an unknown solution could be compared.
Disadvantages of Direct EFP:
a. Minor fluctuations in air or gas pressure cause unstable response in the instrument and lead to errors.
b. Separate analyses and sometimes separate dilutions must be made for sodium and potassium.
c. Mutual excitation – results from the transfer of energy from an excited sodium atom to a potassium atom. The potassium signal is enhanced by the sodium concentration in the specimen.
2. Indirect or Internal Standard EFP – lithium or cesium is added to all calibrators, blanks and unknown in equal concentrations. The flame photometer makes a comparison of the emission of the desired element (Na or K) with the emission of the reference lithium element. By measuring the ratios of emissions in this way, small variations in atomization rates, flame stability and solution viscosity are compensated for. Lithium also acts as radiation buffer to minimize the effects of mutual excitation.
Reasons why lithium is used as a standard:
a. Lithium emission characteristics are sufficiently similar to those of sodium or potassium so that conditions that would produce variations in emission of the test elements would produce similar change in lithium emission.
b. Lithium is normally a trace element in human tissues and does not present interference.
Criteria in choosing the internal standard:
a. Its concentration must be precisely the same in all samples and standards.
b. Energy required of the internal standard must be close to that required to excite the elements being measured.
c. Must not be normally found in solution being analyzed.
Purpose of internal standard in EFP:
To achieve stability when there is fluctuations caused by changes in fuel or air pressure which affects flame temperature and rate of sample aspiration.
C. ATOMIC ABSORPTION SPECTROPHOTOMETRY (AAS)
This analysis is the reverse of flame emission photometry wherein the element is not excited in the flame but instead dissociated from its chemical bonds and placed in an unexcited or ground state (neutral atom). This means that the neutral atom is at low energy level in which it is capable of absorbing radiation at a very narrow bandwidth corresponding to its own line spectrum.
Components of AAS:
1. Hollow cathode lamp as light source – contains argon or neon gas at a pressure of a few millimeters of mercury. An argon–filled lamp produces a blue to purple glow during operation and the neon produces a reddish–orange glow inside the lamp.
Quartz or special glass that allows transmission of the proper wavelength is used as window.
2. Burner – for absorption of energy.
Two types of burner:
a. Total consumption burner – aspirate sample directly into the flame, the gases are passed at high velocity over the end of the capillary suspended in the solution.
Advantages:
(1) Flame is more concentrated and it can be made hotter than other burners, causing molecular dissociation that may be desirable for some chemical systems.
b. Premix burner or laminar flow burner – involves the gravitational feeding of solution through a restricting capillary into an area of high velocity gas flow where small droplets are produced and passed into the flame.
Advantages:
(1) Larger droplets go to waste while the fine mist enters the flame, thus producing a less noisy signal
(2) The pathlength through the flame of the burner is longer than the total consumption burner. This produces a greater absorption and increases the sensitivity of the measurement.
Disadvantage:
(1) Flame is usually not as hot as total consumption burner thus it cannot dissociate certain metal complexes in the flame sufficiently.
e.g. calcium–phosphate complexes
3. Mechanical rotating chopper or nebulizer – modulates the light beam coming from Hollow Cathode Lamp and sprays the sample into the flame.
4. Prism or Diffraction gratings as monochromator (see similar discussions)
5. Photomultiplier (see similar discussions)
6. Meter (see similar discussions)
Flameless AAS – utilizes carbon rod or graphite furnace or strips of tantalum or platinum metal as sample cups in the chamber. The temperature is then raised to dry, char and atomize the sample in the chamber. The atomized element then absorbs energy from corresponding hollow cathode lamp.
Advantages:
1. More sensitive
2. Permits determination of trace metals in small blood samples and tissues.
Interference in AAS and their remedy:
1. Chemical interference – refers to the situation when the flame cannot dissociate the sample into free atoms so that absorption can occur (e.g. calcium–phosphate complexes).
Remedy:
Extraction techniques and the introduction of competing cations to release the element to be measured from complexing or chelating anions.
2. Ionization interference – results when atoms in the flame become excited, instead of only being dissociated and then emits energy of same wavelength that is being measured.
Remedy:
a. Addition of easily ionized substance that will absorb most of the flame energy so that the substance of interest will not become excited.
b. Reduction of flame temperature.
3. Matrix interference may be due to:
a. Enhancement of light absorption by organic solvents.
b. Light absorption caused by formation of solids from sample droplets as the solvent is evaporated in the flame.
c. Refractory oxides of metals formed in the flame.
D. FLUOROMETRY
Flourometry is the determination of the characteristics and amount of luminescence produced by substances when examined under controlled condition.
Fluorometer is distinguished from spectrophotometer by the way the excitation light and emission light are separated into monochromatic light. The flourometer employs interference filters in glass filters to produce monochromatic light for sample excitation and for isolation of fluorescence emission while spectrophotometer uses gratings or prism monochromator for this purpose.
Fluorescence is the property of some chemical compounds to absorb light energy and then reemitting some of this energy in light of a longer wavelength than the light originally absorbed.
Components of Fluorometer:
1. Excitation source
a. Xenon arc lamp – provides energy at the spectral range of 250 – 800 nm. However, the arc between the internal electrodes of the xenon lamp tends to move slightly causing some random variation in the intensity of light reaching the cuvet which is known as arc wandering or flicker.
c. Laser sources:
Properties of laser sources:
(1) Spatial coherence – allows beam diameters in the range of several microns.
(2) Monochromatic light production.
(3) Have pulse widths that vary from microseconds (flash lamp – pulsed lasers) to nanoseconds (nitrogen lasers) to picoseconds or less (mode–locked lasers).
Types of lasers:
(1) Argon ion lasers – used in flow cytometry, has a monochromaticity of 488 nm, spatial coherent and optical alignment easness.
(2) Continuous wave dye lasers – has an excitation wavelength ranges of 260–300 nm, 400–600 nm and 540–900 nm. It has been used in phase–resolved fluorescence.
(3) Helium–Neon (He–Ne) lasers – operates at 633 nm and are typically used for light scattering application such as nephelometric immunoassay and particle size and shape determinations. Newer types has been developed that emit excitation energy at 543 nm (green), 549 nm yellow) and 611 nm (orange).
(4) Helium–cadmium (He–Cd) lasers – emit energy from 5–40 mW of power at 411 nm and from 1 to 10 mW of output at 325 nm.
d. Quartz halogen lamp
e. Mercury arc lamp
2. Excitation and Emission monochromator
a. Intereference filters
(1) All–dielectric multicavity
(2) Hybrid Fabry–Perot coupled–dielectric–layer filter – a filter with metal reflective layers.
· These are combined with sharp cut–off filters to form a single filter package, which removes undesired transmission of higher orders and provides narrow bandwidths, higher peak wavelength transmission and increased band slope. The increased slope of the spectral band makes the transition from peak transmission more abrupt, which is very important for the spectral isolation of excitation and emission bands with a small stokes shift.
Stokes shift – difference between the maximum wavelength of the excitation light and maximum wavelength of emitted fluorescent light.
b. Colored glass filters – used for both excitation and emission wavelength selection but more susceptible to transmitting stray light and they can exhibit unwanted fluorescence.
c. Grating monochromator – device that isolate regions of a spectrum.
3. Sample cell compartment – sets the geometry the instrument use for fluorescence measurements, usually made of fused silica or quartz.
a. Right–angle–detector approach – minimizes background signal that limits analytical sensitivity.
b. End–on approach – allows the adaptation of a fluorescence detector to existing 180–degree absorption instruments. Its sensitivity is limited by the quality of the excitation/emission interference filter pair, the excitation/emission spectral band overlap and the inner filter effect.
c. Front surface approach – provides the greatest linearity over a broad range of concentration because it minimizes the inner filter effect.
4. Photodetectors:
a. Photomultiplier (PM) tubes – gives a wide choice of spectral responses, rapid photon response time (i.e., nanosecond response time) and sensitivity (due to possible gain of 106 electrons at the anode of the PM tube for each incident photon hitting the photo cathode).
b. Charge–coupled detectors – multichannel devices that have good dynamic range and a signal–to–noise ratio that is superior to PM tubes. Utilizes mercury lamp as light source.
Types of Fluorometer:
1. Time–released fluorometer – the sample is illuminated with an intense brief pulse of light and the intensity of the resulting fluorescence emission is measured as a function of time with a fast detector system.
2. Phase–resolved fluorometers – a continuous–wave laser illuminates the sample and the fluorescence emission response is monitored for impulse and frequency.
3. Fluorescence polarization – used to quantitate analytes such as therapeutic drugs by the use of change in fluorescence depolarization following immunological reactions.
4. Hematofluorometer – is a single channel front surface photofluormeter dedicated to the analysis of zinc protophorphyrin in whole blood. A typical hematofluormeter uses a quartz tungsten lamp, a narrow bandpass excitation filter (420 nm), front surface optics, a narrow bandpass filter (594 nm) and PM tube.
5. Laser–induced fluorometer – records the passage of individual fluorescent or fluorescent–labeled molecules, cells or particles through a flow cell cuvet on which a laser beam is focused. The use of laser–induced fluorescence to detect a small number of molecules in a flowing stream is important in the application of flow cytometry, flow injection analysis, high performance liquid chromatography and capillary zone electrophoresis.
An argon ion laser is used for excitation energy and its beam focused tightly through a sheath–flow capillary flow cell. Fluorescence is collected through a microscope objective and 580 nm bandpass filter prior to detection with a PM tube. An amplifier discriminator is used to adjust the signal counts to minimize background interference. The resulting sample counts are processed through a multichannel scaler and stored for data analysis by a computer.
6. Flow cytometry – a combination of laser–induced fluorometry and particle light scattering analysis capable of measuring multiple parameters, including cell size (forward scatter), granularity (90–degree scatter), DNA content, RNA content, DNA A++/G+C nucleotide ratios, chromatin structure, antigens, total protein content, cell receptors, membrane potential and calcium ion concentration as a function of pH.
The multiple parameters stated above are used in:
a. Hematology & immunology
(1) T–cell subsets
(2) Tissue typing
(3) Lymphocyte stimulation
(4) Antigen–antibody reaction
b. Oncology
(1) Diagnosis, prognosis and treatment monitoring
c. Microbiology
(1) Bacterial identification
(2) Antibiotic sensitivity
d. Genetics
(1) Karyotyping
(2) Carrier state detection
e. Parasitology
f. Reproduction and fertility studies
g. Virology
h. Cervical cytology
Factors that influence fluorescence measurements:
1. Concentration effects
Inner filter effects is caused by a loss of excitation intensity across the cuvet path length as the excitation light is absorbed by the fluorophor. Thus, as the fluorophor becomes more concentrated, the absorbance of the excitation intensity increases, and the loss of the excitation light as it travels through cuvet increases. This effect is most often encountered with a right–angle fluorescence instrument in which the emission slits are set to monitor the center of the sample cell where the absorbance of excitation light is greater than at the front surface of the cuvet.
Concentration quenching occurs when a macromolecule, such as an antibody, is heavily labeled with fluorophor such as fluorescein isothiocyanate (FITC). When this compound is excited, the fluorescence labels are so spatially close that radiation less energy transfer can occur. Thus, the resulting fluorescence is much lower than expected for the concentration of the label. This is a common problem in flow cytometry and laser–induced fluorescence when attempting to enhance detection sensitivity by increasing the density of the fluorescing label.
2. Background Effects
Rayleigh scattering is the result of an elastic collision of light with a molecule; the molecules rotational and vibrational energy is changed but because the collision is elastic, no energy is given or taken up. With no energy change, the light emitted occurs with no change in wavelength.
Raman effect is the result of inelastic collision where energy is lost or taken up by the molecule.
3. Solvent Effects
Quenching is related to the interaction of the fluorophor with the solvent or with a solute dissolved in the solvent. Such interaction results in a loss of fluorescence owing to energy transfer or other mechanisms but there is no effect on the absorbance spectrum of the fluorophor. An example of quenching is the loss of fluorescence when halides are added to quinine in dilute sulfuric acid.
4. Sample Matrix Effects
Serum or urine samples contains several compounds capable of giving fluoresce giving an unwanted background fluorescence for the determination.
5. Temperature Effects
Fluorescence intensity decreases with increasing temperature by approximately 1–5% per degree Celsius. Furthermore, collisional quenching decreases with increasing viscosity, thus reducing quenching of fluorescence. Therefore, fluorescence intensity can be enhanced by either increasing reaction viscosity or lowering solvent temperature. Temperature effects can be minimized by controlling reaction temperature and warming samples or reagents, or both, if they have been refrigerated.
6. Photodecomposition
This can be prevented by:
a. Always use the longest feasible wavelength for excitation that does not introduce light scattering effects.
b.Decrease the duration of excitation of the sample by measuring the fluorescence intensity immediately after excitation.
c.Protect unstable solutions from ambient light by storing them in dark bottles.
d.Remove dissolved oxygen from the solution.
E.TURBIDIMETRY
Turbidimetric measurement determines the amount of light blocked by a particulate matter as light passes through a cuvet. A light blocked by a suspensions of particles in a cuvet depends on:
a. Number of particles present
b. Cross sectional area of each particle
c. Depth of tube
Problems in turbidimetric analysis:
a. Particle size of standard may not be the same as that of the sample.
b. The need to keep the length of time between sample preparation and measurement as constant as possible.
c. Particles may settle down while measurements are being done thus producing an error. This can be controlled by using gum arabic or gelatin which provides viscous medium that retards the setting of particles.
Light should not fall on the photometer used for this test because errors in instrument will augment other errors in the test. Turbidimetric methods are used in some liver function tests like thymol turbidity test and zinc turbidity test.
F. NEPHELOMETRY
Nephelometric measurement determines the amount of light scattered by the small particles or colloids in the sample cuvet.
Variables to control this method are:
a. Number of the particles
b. Size of the particles
c. Wavelength of the incident light
An advantage over turbidimetry is that nephelometric measurements are capable of greater precision. Both are not inherently used because they are incapable of high precision. Nephelometry is used in the determination of lipoproteins and triglycerides.
G. SCINTILLATION COUNTER
This is used to measure the disintegration per minute of time of a radioisotope.
Types of radiation:
1. Alpha – positively charged particles, resembles the nucleus of a Helium atom with mass of 4.
– have very little energy.
2. Beta – resembles electron with negatively or positively (B– and B+) charge but essentially has no mass.
– exist in two forms: soft and hard beta
e.g. 14C, 3H, 32P
3. Gamma – a form of electromagnetic energy which resembles X–rays and with no mass, only energy.
– exist in two forms: soft and hard gamma.
e.g. 125–Iodine, 131–Iodine, 60–Cobalt
Use of radioisotope in Medical Diagnostic Procedures:
1. As label – RIA
2. As locator – in imaging or scanning
3. As tracer – in scanning
Types of Scintillation counter:
1. Solid scintillation counter – measures gamma radiation using Thallium activated NaI crystal as scintillator and PM tube as detector with preamplifier circuit.
2. Liquid scintillation counter – measures beta radiation using liquid fluor as scintillator.
ELECTROCHEMISTRY
Electrochemistry is concerned with chemical transformations that involve proton or electron tranfer and result in the flow of electricity. Analytical electrochemistry for the clinical laboratory includes potentiometry, amperometry, coulometry, voltammetry, conductometry and polarography. Generally, amperometry and potentiometry are the most commonly used technique and these centers on blood gas measurements.
A.POTENTIOMETRY
Potentiometry is the measurement of the electrical potential difference between two electrodes in an electrochemical cell. An electrochemical or galvanic cell always consists of two electrodes (electron or metallic conductors) that are connected by an electrolyte solution (ion conductor). An electrode or half cell consists of a single metallic conductor that is in contact with an electrolyte solution. The two electrodes for potentiometric measurements are the reference electrode (left electrode, ML) and the indicator or measuring electrode (right electrode, MR).
The electromotive force (E or EMF) is defined as the maximum difference in potential between the two electrodes (right minus left) obtained when the cell current is zero.
Both an indicator and a reference electrode are necessary to measure the potential (E) of a solution. The potential of the indicator electrode can be made to respond proportionally to the concentration of the substance of interest, while the reference electrode must maintain a constant voltage under controlled conditions for a significant length of time.
In potentiometry, the potential is measured and the relationship between the measured voltage and the sought for concentration is shown by the Nernst equation:
E = Eo + 0.059 log [Cox]
n [Cred]
where:
E – the potential measured at 25oC
Eo – the standard reduction potential
n – the number or electrons involved in the reaction
Cox – the molar concentration of the oxidized reaction form
Cred – the molar concentration of the reduced reaction form
Types of potentiometry:
1. Indirect potentiometry – involves the determination of the concentration or total ion (free and bound) diluted with a suitable diluent that liberates the complex–bound ion from its binding agent. The dilution should at the same time serve as to establish a constant ionic strength, so that a constant activity coefficient is obtained independently of variations in ionic strength of the original sample.
2. Direct potentiometry – involves the determination of the concentration of free unbound ion or more precisely the activity of the ion with the use of undiluted sample.
e.g. Calcium determination
3. Titration utilizing potentiometric end–point detection – electrode is only use to sense the sudden change in activity as the end–point is reached. Although more labor–intensive, this technique is generally considered among the most accurate and precise analytical method available.
Components of a potentiometer:
1. Reference electrode – consists of internal wire, filling solution and a permeable outer casting.
a. Inert metal electrodes immersed in solutions containing redox couples.
(1) Platinum and gold – used to record the potential of a redox couple dissolved in an electrolyte solution. However, the presence of a catalyst is required to establish a reproducible potential. The catalyst can be a small amount of another redox couple, a so called “mediator” that readily equilibrates with both the metal electrode and the more sluggish redox couple of interest. Examples of mediators are methylene blue and quinhydrone.
(2) Normal Hydrogen Electrode (NHE) – consists of a platinized platinum electrode in a 1.228 N HCl solution with hydrogen at atmospheric pressure bubbled over the platinum surface.
(3) Quinhydrone electrode – consists of a platinum or gold electrode immersed in a saturated solution of quinhydrone, which is equimolar mixture of quinone and hydroxyquinone.
· The hydrogen electrode and the quinhydrone electrode have both been replaced by the glass electrode for routine pH measurements, although the use of quinhydrone electrode has been proposed in connection with a pCO2 electrode.
b. Metal electrodes whose metal functions as a member of the redox couple
(1) Saturated calomel electrode (SCE) – consists of mercury covered by a layer of calomel which is in contact with an electrolyte solution containing chloride.
(2) Silver–silver chloride electrodes – consists of silver wire, electrolytically coated with silver chloride, which dips into a solution containing chloride ions. It is used in the direct measurement of chloride activity in serum.
2. Indicator electrode
a. Platinum wire
b. Planar surface of any metal
c. Carbon rod
d. Mercury
THE ION SELECTIVE ELECTRODE (ISE)
The ion selective electrode is carried out using an electrode designed specifically for the detection of the component of interest. Selectivity is created in part by the use of porous organic polymer which allows only one ion of a specific size to pass through. Other ions which might alter the potential gradient are excluded from the system because their ionic radius is different from that of the ion being selected. A wide variety of polymeric materials are employed in these electrodes, either as simple membranes to separate ions or as part of an ion complexing, liquid membrane system.
Complexing agents are added to bind ion of interests and immobilizes it inside the electrode. This binding and trapping diminishes the tendency o the ion to move back to the outside of the electrode membrane, which would decrease the degree of charge separation and lower the potential difference.
Enzyme–coupled, ion selective electrode has been developed to measure enzyme, carbon dioxide or glucose directly or indirectly. The major drawbacks to widespread use of enzyme electrodes are slow response time and lack of long–term stability. An enzyme reaction used to take place before the product can be detected by the electrode.
One of the disadvantage of use of ISE is the presence of high concentrations of lipids and proteins found in serum or plasma which changes the actual water volume of the sample and can give rise to erroneous results. Furthermore, proteins can coat the electrode and decrease the amount of diffusion.
Design of membrane electrode:
1. Flat or bulb–shaped
2. Inverted flat or bulb–shaped
3. Capillary type
4. Flow Through
5. Catheter Tip
6. Glass Capillary
Classification of ISE:
1. Glass electrodes – made from glass consisting of melt of silicon dioxide with added oxides of various metals. Membranes prepared with directed selectivity for H+, Na+, K+, Li+, Rb+, Cs+, Ag+ and NH4+ are also available.
a. H+ selective glass electrode – consists of silicon dioxide, sodium oxide and calcium oxide in the molar ratio of 72.2: 21.4: 6.4, respectively. Newer type consists of silicon dioxide, lithium oxide and calcium oxide in the ratio of 68: 25: 7, respectively. It is bulb–shaped for most titration purposes and flat for surface measurements, but it has an inverted bulb–shaped when used for microanalysis. For pH measurements in blood, the thermostatted capillary glass electrode has been useful.
b. Na+ selective glass electrode – consists of silicon dioxide, sodium oxide and aluminum oxide in a proportion of 71: 11: 18. Lithium aluminum silicates are also found suitable. Electrodes with a flat surface have been used for the direct measurement of Na+ activity on the skin surface for the diagnosis of cystic fibrosis. Capillary electrodes are used for Na+ in serum.
c. K+ selective glass electrode
d. Li+ selective glass electrode
e. NH4+ selective glass electrode
2. Solid State Electrodes – a homogenous membrane consists of single crystal or heterogenous membrane that consist of an active substance embedded in an inert matrix. This include europium–doped lathanum fluoride crystal, silver chloride, silver bromide, silver iodide, silver sulfide, cupric selenide and others.
The silver chloride membrane electrode is used for measurement of the activity of chloride in sweat by direct measurement of the skin surface. The fluoride electrode has been utilized for measurements of the fluoride concentration in blood, urine and saliva as well as in bone and tooth enamel after dissolution.
3. Liquid ion exchange electrode – consist of an inert solvent in which an ion selective carrier substance is dissolved. Both solvent and carrier should be insoluble in water. The membrane solution can be separated from the test solution by means of a collodion membrane or a porous matrix can be impregnated with the membrane solution. The inert solvent (plasticizer) and the ion selective carrier are often embedded in a matrix of polyvinylvhloride (PVC) obtained when solutions of PVC in tetrahydrofuran are evaporated into thin semi–solid membranes.
a. K+ selective membranes – done by dissolving valinomycin antibiotic in a suitable solvent. Valinomycin is a neutral carrier that binds K+ in the center of a ring of oxygen atoms.
b. NH4+ selective membrane – based on a mixture of the antibiotics nonactin and monactin, which are also neutral carriers.
c. Ca++ selective membrane – can be made by dissolving the calcium salt Ca2+–bis (di–p–octyl–phenyl phosphate) in PVC. Two di–p–octyl phenyl phosphate ions bind calcium ion to act as the Ca2+ carrier. Dioctyl phenyl phosphonate is added as a plasticizer to the PVC.
4. Gas electrode – for measurement of a specific gases in gas mixtures or in solution. This is separated from the test solution by a thin, hydrophobic gas permeable membrane (e.g. polyethylene, polypropylene, microporous teflon, silicone rubber), but separation may also simply be a small “air gap.”
a. Carbon dioxide electrode – the sample in this case is in contact with a membrane that is permeable to glass electrode by a thin film of bicarbonate solution (5 mmol/L). The CO2 gas diffuses from the sample (or test gas) through the membrane and rapidly enters into equilibrium with the bicarbonate solution, thus altering its pH. The pH of the bicarbonate solution is a simple function of pCO2.
The CO2 electrode has been used extensively for measurements in arterial blood samples. More recently its application has been extended to transcutaneous measurements.
b. Ammonia electrode – same as CO2 electrode except that the bicarbonate solution is replaced with an ammonium chloride solution.
Objections in potentiometric measurements:
1. Protein interference
Cause: protein free and protein containing solutions
Remedy: use of dialysis membrane
Disadvantage: prolongation of response time of the electrode from less than 1 second to greater than 20 seconds for 98% response.
2. Liquid–liquid junction potential
Cause: direct contact of two solutions of different ionic composition.
Remedy: use of concentrated potassium chloride solution as the bridge solution.
B.AMPEROMETRY
Amperometry is the measurement of the current flowing through an electrochemical cell when a constant potential is applied to the electrodes.
Applications of amperometry:
1. Clark electrode (pO2 electrode)
a. Parts:
(1) Silver/silver chloride anode in phosphate buffer with added potassium chloride.
(2) Platinum cathode covered by a gas–permeable membrane (e.g. propylene).
(a) To prevent proteins and other (dissolved)oxidants from gaining access to the cathode surface.
(b) To limit the diffusion zone to the membrane and, hence, prevent variations in the diffusion coefficient of oxygen in the test solution (or gas) from influencing the result.
b. Uses:
(1) Measurement of the concentration of total oxygen in the blood after liberation of hemoglobin–bound oxygen with ferricyanide or carbon monoxide (by forming methemoglobin and carboxyhemoglobin, respectively).
(2) Transducer in a broad range of oxidase–based enzyme electrode.
C. COULOMETRY
Coulometry is the technique used to measure the amount of electricity passing between two electrodes in an electrochemical cell. The amount of electricity is directly proportional to the amount of substance produced or consumed by the redox process at the electrodes. This is called the Faraday’s law and can be expressed as :
Q = Z x n x F
Where:
Q – amount of electricity (in Coulomb–ampere second)
Z – numerical stoichiometric number of electrons involved in the
reduction or oxidation reaction (in liter).
n – amount of substance reduced or oxidized (in mol).
F – Faraday constant (96487 C–mol– 1)
Application:
1. Coulometric titration of chloride using Cotlove Titration in which silver are generated by electrolysis from is silver wire used as the anode.
2. Acid–base titration using platinum generator electrode separated by a sintered glass filter.
D. VOLTAMMETRY
Voltammetry are used to study solution composition based on the current–potential relationships obtained when potential of an electrochemical cell is varied. The foremost analytical advantage of voltammetric methods are sensitivity and the capability for multielement and speciation studies.
Parts:
1. Working electrode
2. Reference electrode
3. Counter or Auxillary electrode
Types:
1. Linear potential sweep
2. Potential step
3. Anodic and cathode stripping voltammetry
4. Phase sensitive AC voltammetry
E.POLAROGRAPHY
Polarography consist of gradual application of increasing voltage and simultaneous measurement of current. It involves a two electrode system, consisting of a reference electrode and indicator electrode. The indicator electrode frequently consists of a thin ribbon of mercury metal flowing slowly through a small gas capillary and dropping into the solution to be measured. Generally, linearly increasing cathodic voltage is applied to the dropping mercury electrode (DME) and the current that flows between the two electrodes is measured and is proportional to the concentration of the substance of interest. Polarography is used to trace metals, oxygen, Vitamin C and amino acid concentration.
F. CONDUCTOMETRY
Conductometry is the measurement of the current flow between two non–polarized electrodes and between which a known electrical potential is established.
Electrolytic conductance is a measure of the ability of a solution of electrolytes to carry an electrical current by the migration of ions under the influence of a potential gradient.
Factors in the rate of migration of ions:
1. Ionic charge and size
2. Solvent viscosity
3. Magnitude and frequency of applied potential
Uses of conductometry:
1. To monitor the performance of deionizers and to indicate whether the ion exchange resin should be regenerated.
2. For end–point detection in titration.
Application of conductometry:
The Coulter principle for electronic counting of blood cell in suspensions relies on the fact that the conductivity of blood cells is lower than that of a salt solution employed as suspension medium. The cell suspension is forced to flow through a tiny orifice. Two electrodes are placed on either side of the orifice and a constant current is established between the electrodes. Each time a cell passes through the orifice, the resistance increases, this causes a spike on the electrical potential difference between the electrode. The pulses are then amplified and counted.
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