OSMOMETRY
Osmometry is a technique for measuring the concentration of a solute particle which, in turn, is related to the osmotic pressure of a solution. The osmotic pressure governs the movement of water across membranes separating two different solutions. Osmotic pressure can be defined as the pressure that is required to maintain equilibrium between the solution on the one side of the membrane and the water on the other side of the membrane.
One osmol of the substance is equal to the gram molecular weight divided by the number of particles or ions into which the substances dissociate in solution.
Colligative properties of solutions:
2. Vapor pressure depression
1. Vapor pressure osmometer – osmolality measurement is not related directly to a change in vapor pressure but to the decrease in the dew point temperature of the pure solvent caused by the decreased in vapor pressure of the solvent by solutes.
2. Colloid osmotic pressure osmometer (COP) – directly measures the contribution of macromolecules to the osmolality.
3. Freezing point depression osmometer or Cryoscope – used to determine the concentration of osmotically active particles in physiological fluids because its measurement is simple and convenient.
Components of an osmometer:
1. Thermostatically controlled cooling bath or block maintained at –7oC.
2. Rapid stir mechanism to initiate (“seed”) freezing sample.
3. Thermistor – a glass bead attached to a metal stem whose substance varies rapidly and predictably with temperature. It is connected to a wheat stone bridge circuit to measure the temperature of the sample.
4. Galvanometer – displays the freezing curve and that is used as a guide when the potentiometer is used.
5. Potentiometer – to null the current in the wheat stone circuit.
· In some automated instruments, components 4 and 5 are replaced by a light emitting diode (LED) display that both indicates the time course of the freezing curve and locks in the final result in a numerical display.
GASOMETRY
Gasometry is the measurement of the amount of gas liberated in chemical reaction. The gas is liberated in chemical reaction. The gas is liberated from the blood by an appropriate reagent and is extracted by shaking under a partial vacuum and the gas liberated is measured.
The measurements of gases by volumetric and manometric methods have been done on carbon dioxide and oxygen determinations. At present, oxygen is frequently measured by PO2 electrodes.
Gas laws:
a. Boyle’s Law – at constant temperature, a fixed weight of gas occupies a volume inversely proportional to the pressure exerted upon it.
V1 = P2
V2 P1
where: V1 – initial volume P1 – initial pressure
V2 – final volume P2 – final pressure
This is the basis of the manometric blood gas measurements.
b. Charles Law – at constant pressure, the volume occupied by a fixed weight of gas is directly proportional to the temperature.
V1 = T1
V2 T2
where: V1 – initial volume V2 – final volume
T1 – initial temperature T2 – final temperature
Charles Law is the basis of the volumetric method of gas analysis.
ELECTROPHORESIS
Electrophoresis refers to the migration of charged solutes or particles in a liquid medium under the influence of an electrical field.
Terms associated with electrophoresis:
1. Iontophoresis – migration of small ions.
2. Isotachophoresis – a technique in which sample components ultimately separate into adjacent zones that all migrate at the same rate.
3. Isoelectric point (pI) – pH where net charge on molecule is zero.
4. Ampholytes or zwitterion – a molecule which can either be positively or negatively charged.
5. Anode – a positive electrode where negative charge particles (usually acidic) migrate.
6. Cathode – a negative electrode where positive charge particles (usually basic) migrate.
The most common examination subjected to electrophoresis are serum proteins (applicable to CSF too). The proteins in serum when subjected to electrophoresis will show different migration rates because they are different with regards to electrical charges, size and shape.
Two major types of electrophoresis:
1. Free electrophoresis – the electrical field is applied directly to a solution.
2. Zone electrophoresis – the charged particles are placed in a stabilizing medium which will contain the protein after migration. The proteins in the stabilizing medium can be stained and examined later on.
Types of stabilizing media used in electrophoresis:
a. Filter paper
b. Cellular acetate
c. Layer of starch granules
d. Agar gel
e. Starch gel
f. Acrylamide gel
Two major conditions for selection of type of media to another one:
a. Time required for separation
b. Degree of resolution
Components of electrophoresis system:
1. Support medium – site of electrophoretic separation; hold the sample and provide a path for migration of proteins; act either as sieve (separation on basis of size) or charge in system (separation on basis of net electric charge).
e.g. agarose, cellulose acetate, etc.
2. Electrophoresis chamber
3. Power supply – converts alternating line current for the operation of the system.
Procedure for electrophoresis:
1. A hydrated support material such as freshly prepared agarose gel or previously wetted cellulose acetate is placed into the electrophoresis chamber.
2. Sample is applied to the support and electrophoresis is conducted for a determined length of time using either constant voltage or constant current.
3. The support is then removed from the electrophoresis cell and rapidly dried or placed in a fixative to prevent diffusion of sample components.
4. It is then treated with a dye–fixative reagent to locate and visualize the individual protein zones by staining.
5. After washing out excess dye, the support is dried or placed in clearing agent.
6. Quantitation of sample either by elution method or by densitometry.
Methods of quantitating electrophoretic samples:
1. Densitometry – measurement of amount of light passing through the fractions.
2. Elution method – involves cutting the support into zones containing the individual fractions and eluting the adsorbed dye by means of suitable solvents such as basic buffers, weak alkali (0.1 mol/L NaOH) or alcohol solutions.
Commonly employed stains in electrophoresis and their suggested wavelength for quantitation:
Densitometry Elution
Staining of serum proteins
Amido Black (Napthol
Blue Black) 640 600
Blue Black) 640 600
Bromphenol Blue 600 595
Coomasie Brilliant Blue
G–250 (Brilliant Blue G) 595 610
Coomasie Brilliant Blue
R–250 (Brilliant Blue R) 560 580
Nigrosin 540 560
Ponceau S 520 565 (alkaline)
520 (neutral)
Staining of isoenzymes
Nitrotetrazolium Blue
(as the formazan) 570 –
Staining of lipoproteins
Fat Red 7B (Sudan Red 7B) 540 –
Oil Red O 520 –
Sudan Black B 600 –
Types of electrophoresis:
1. Agarose Gel Electrophoresis (AGE)
Use in the analysis of serum proteins, hemoglobin variants, lactate dehydrogenase isoenzymes, lipoprotein fraction and other substances.
An agar is a polyacrylamide mixture derived from seaweed which is composed of two fractions:
a. Agaropectin – contains acid sulfate and carboxylic acid groups and accounts for the considerable endosmosis and background staining that are observed with unfractionated agar.
b. Agarose – the free of ionizable groups portion.
Advantages:
a. Electric neutrality – there is minimal electric charge on the agarose polymer itself, so there is little or no interaction with protein expected.
b. Separation is strictly on the basis of electric charge.
c. Greater uniformity of materials and better reproducibility pattern.
d. Amount of sample needed: 0.6 – 3.0 ul
e. Running time: 30 – 90 minutes
2. Cellulose Acetate Electrophoresis (CAE)
Reaction of the hydroxyl groups of cellulose with acetic anhydride will acetylate to form the raw material for cellulose acetate membranes.
Amount of sample needed: 0.3 – 2.0 ul
Running time: 20 minutes – 1 hour
Advantage:
Ability to store the transparent membranes for long periods.
3. Polyacrylamide Gel Electrophoresis (PAGE)
Cellulose acetate and agarose when used as support media yields five protein fractions namely: albumin, alpha–1, alpha–2, beta and gamma globulins, however, with the use of PAGE, it can yield more than 20 protein fractions which is important in the study of genetic variants and isoenzymes.
PAGE utilizes layers of gel that differ in composition and pore size.
Procedure:
a. The individual gels are first prepared in sites in glass tubes by polymerizing a gel monomer and a cross–linking agent with the aid of an appropriate catalyst.
b. The first gel to be poured into the tubular shaped electrophoresis cell is the small pore separation gel.
c. After 30 minutes, during which gelatin takes place, a large pore gel, the spacer gel, is cast on top of the separation gel.
d. Then a large pore monomer solution containing a small amount of serum (3 ul) is polymerized above the spacer gel so that the finished product is composed of three different layers of gel.
e. When electrophoresis begins, all protein ions migrate through the large pore gels and stack up on the separation gel in a very thin zone. This process serves to concentrate protein components at the border zone so that preconcentration of specimens with low protein content (e.g. CSF) may not be necessary.
f. Separation of the individual protein ions then takes place in the bottom separation gel, not only on the basis of their charge but also on the basis of molecular size.
Advantages:
a. Acrylamide gel is thermostable, transparent, strong and relatively chemically inert and can be made in a wide range of pore sizes.
b. The gel is unchanged, thus eliminating electroendosmosis.
4. Starch Gel Electrophoresis
Separates macromolecular ions on the basis of both surface charge and molecular size. This can be accomplished both in a horizontal or vertical position. Starch gel is used in a concentration of 10 to 16 g/dl. The pH of the buffer is generally chosen to be between 8.6 and 9.0, but may range between 3 and 11.
New approaches to electrophoresis:
1. Isoelectric focusing (IEF)
A technique that separates amphoteric compounds by virtue of migration in a medium possessing a stable pH gradient with pH varying in the direction of the migration. The pH gradient is created by the use of amphoteric polyaminocarboxylic acids (carrier ampholytes), a group of compounds with molecular weights of 300 to 1000. IEF has been adapted both in AGE and CAE.
2. Two–dimensional (2–D) electrophoresis
A separation technique that uses a charge–dependent IEF electrophoresis in the first dimension and a molecular weight dependent electrophoresis in the second dimension. The first dimensional electrophoresis is carried out in a large pore medium such as agarose gel or large–pore polyacrylamide gel. Ampholytes are added to yield a pH gradient. The second dimension is often polyacrylamide in a linear or gradient format.
The 2–D electrophoresis method of O’ Farell uses polyacrylamide gel isoelectric focusing in 130 x 2.5 mm (ID) tubes for the first dimension and incorporates ampholytes that cover a pH range of 3 – 10 units.
In the O’Farrell method, SDS is used in the second dimension, B–mercaptoethanol in the first. Other authors use SDS in both dimensions as well as in sample preparation. The use of B–mercaptoethanol and SDS serves to denature proteins to polypeptide by reducing disulfide bonds and depolymerizing proteins.
3. Capillary electrophoresis
A method of separation which is a combination of zone electrophoresis, isotachophoresis, isoelectric focusing and gel electrophoresis carried out in a small bore (25–75 um) fused silica capillary approximately 100 cm in length. Instrumental advantages of capillary electrophoresis include the ease of automating sample introduction and the possible application of a wide variety of detectors.
Capillary electrokinetic chromatography combines differences in the electrophoretic mobililty of solutes with partitioning between a moving aqueous phase and a slower–moving unicellar phase.
Problems encountered with electrophoresis:
1. Buffer concentration
Buffer ions has two purpose in electrophoresis: they carry the applied current and they fix the pH at which electrophoresis is carried out and thus, determine the kind of electrical charge on the solute and the extent of ionization of the solute. Increase ionic strength increases mobility.
Commonly used buffer:
a. Barbital buffer
b. Tris–boric acid–EDTA buffer
2. Electroendosmosis
Zeta potential on electrokinetic potential is the potential which exist between the fixed ions and the associated cloud of ions. When a current is applied to such a system, charges attached to the immobile support remain fixed, but the cloud of ions in solution is free to move to the electrode of oppose polarity. Since the ions in solution are highly hydrated, this movement of the ionic cloud results inmovement of the solvent as well. This movement of solvent of its solutes relative to the fixed support is referred to as endosmosis.
Factors affecting electrophoresis:
1. Net electric charge of the molecule
2. Size and shape of the molecule
3. Electric field strength
4. Nature of supporting media
5. Temperature of operation
Precautions in electrophoresis:
1. Buffer should be refrigerated when not in use.
2. Cold buffer is preferred because resolution is improved and evaporation from the electrophoretic support is lessened.
3. Buffers used in small volume apparatus should be discarded after each run because of pH changes resulting from the electrolysis of water that accompanies electrophoresis.
4. A stain solution of 100 ml maybe used for a combined total of 60 in2 of cellulose acetate or agarose film.
5. The stain solution maybe considered faulty if leaching of stained protein zones occurs in the 5% acetic acid wash solution or in the clearing solution.
6. Stain solution must be stored tightly covered to avoid evaporation.
Problem solving in electrophoresis:
1. Discontinuities in sample application maybe due to dirty applicators. Use caution in clearing these applicator because they are easily bent. Twin–wire applicators are best cleaned merely by dipping in water with agitation followed by removal of water and residue by gently pressing the applicators against absorbent paper. It is inadvisable to clean wires by manual wiping.
2. Distorted protein zones maybe due to:
a. Bent applicators or too excessive drying of portions of the electrophoretic support.
b. Over application
c. Excessive drying
d. Improper tension
3. Irregularities other than the broken zones in sample application probably are due to excessively wet cellulose acetate films.
4. Unusual bands are artifacts that maybe easily recognized. Hemolyzed samples are frequent causes of an increased b–globulin (where free hemoglobin migrates), or an unusual band between the alpha–2 and beta–2 globulin maybe the result of a hemoglobin–haptoglobin complex. A band occurring at the starting point of an electrophoretogram maybe fibrinogen. The sample should be verified as being serum before this band is reported as an abnormal protein.
5. Artifacts are recognized by their lack of regular somewhat diffuse appearance that proteins normally show: it is actually denatured protein resulting from a deteriorated serum or from damage done to the cellulose acetate by twin wire applicator.
CHROMATOGRAPHY
Chromatography is used to separate or purify small amounts of closely related compound from one another in a mixture. This is based on one or more of the four physicochemical principles of absorption, partition, ion–exchange and exclusion (molecular sieving).
A chromatographic system consist of a mixture of phases:
1. Mobile phase – a liquid or gas moves through the system.
2. Stationary phase – a liquid or solid is fixed or motionless during the process of chromatography.
In ascending chromatography, the movement is upward while in descending chromatography, the movement is downward.
A classification can be made depending upon whether the stationary phase is solid or liquid. If it is solid, the method is termed absorption chromatography; if it is a liquid, the method is partition chromatography.
Separation mechanism in chromatography:
1. Ion exchange chromatography – the differences in the sign and magnitude of ionic charges are the basis for separation. This technique is most useful for separation of inorganic ions, amino acids, nucleotides and proteins.
Types of material used for separation:
a. Cation–exchange resins – characterized by the presence of negatively charged groups.
(1) Strongly acidic groups: e.g. sulfonate ions
(2) Weakly acidic groups:
e.g. carboxylate ions sulfomethyl (SM)
carboxymethyl (CM) sulfoethyl (SE)
phosphate (P) sulfopropyl (SP)
b. Anion–exchange resins – characterized by the presence of positively charged groups.
(1) Strongly basic quarternary amines:
e.g. triethylaminoethyl
(2) Weakly basic groups:
e.g. aminoethyl (AE)
diethylaminoethyl (DEAE)
epichlorohydrin–triethanolamine (EC–TEOLA)
guanidoethyl (GE)
Clinical application:
a. Removal of ions in water (deionization)
b. Chromatographic separation of hemoglobin variants
c. Isoenzymes of creatinine kinase and lactate dehydrogenase and amino acids
d. Removal of interfering ions
2. Steric Exclusion chromatography – depends primarily on molecular size selectivity to separate molecules, although molecular shape and hydration for exert effects.
Synonyms: Gel filtration
Gel permeation
Size exclusion
Molecular sieve chromatography
Stationary phase material used:
a. Dextran (Sephadex)
b. Polyacrylamide (Bio–Gel)
c. Agarose (Sepharose)
d. Polysterene–divinylbenzene
e. Porous glass
Hydrophilic gels – have been designed to be used in water for the separation of polysaccharides as well as of enzymes, antibodies and other proteins.
Hydrophobic gels – for separation of non–polar species such as triglycerides in non–aqueous mobile phase.
3. Adsorption chromatography – separation based on the electrostatic, hydrogen–bonding or dispersive interactions between a molecule and solid support or adsorbent.
a. Gas chromatography – used to separate low–molecular weight compounds and compounds that are normally gases at room temperature.
e.g. methyl, ethyl, isopropyl alcohol
media employed: “molecular sieves”
porapak polymers
alumina
b. Liquid chromatography – uses three types of adsorbents:
(1) Non–polar adsorbents: charcoal and divinyl benzene
(2) Acidic polar adsorbents: silica gel
(3) Basic polar adsorbents: silanol & florisil
c. Liquid–solid chromatography – separation based on competition between the mobile phase component and the solutes for the adsorption sites on the support.
Disadvantages:
a. Preparation of a support with a homogenous distribution of adsorption sites.
4. Partition chromatography – separation based on the relative solubility of the solute molecule in the stationary and mobile phase. Eluotropic series can be developed for partition chromatography using the Hildebrand solubility parameters or other solubility treatments.
a. Normal–phase partition chromatography – a polar solvent such as b,b’–oxydipropionitride is used as the stationary phase, whereas a non–polar solvent mixture such as hexane and ethanol is used as the mobile phase.
e.g. Separation of fatty acids with squalene stationary phase
Using a mixture of water and acetonitrile as the eluent
b. Reversed phase partition chromatography – a separation wherein the stationary phase is less polar than the mobile phase.
5. Affinity chromatography – separation based on interaction that occurs between biochemical species: specific enzyme–substrate, hormone–receptor or antigen–antibody complexes.
Synonyms: Hydrophobic chromatography
Covalent chromatography
Metal–chelate chromatography
Template chromatography
Charge–transfer chromatography
Support media: agarose, cross linked dextrans, polyacrylamide, cellulose, polysterene and controlled–pore glass
The stationary phase is prepared by immobilizing a molecule called a ligand because it participates in binding in a support either directly or via a spacer. If the interaction between analyte and ligand is specific. The analyte maybe displaced in a single step by addition of substrate or an inhibitor or alternatively by a pH change, by an ionic change or by addition of a hydrogen bond–breaking agent or chaotropic agents such as guanine hydrochloride, urea or sulfite.
Forms of chromatography:
1. Planar chromatography – mixtures are separated on a planar surface of the stationary phase.
a. Paper chromatography – the stationary phase is a layer of water or a polar solvent coated onto the paper fibers. The type of paper used is selected based on its homogeneity, wet strength, thickness and level of impurities and on the migration rate of mobile phase through it.
b. Thin–layer chromatography (TLC) – a thin layer of the support particles is spread on a flat plate of glass or plastic. Using a capillary tube, syringe or mechanical applicator, aliquots of samples are applied on the plate along a line that is parallel to the bottom edge of the plate and a few centimeters above it.
(1) Ascending technique – the plate is then placed into a tank containing a small volume of mobile phase such that the surface of the mobile phase is located below the applied spots. The mobile phase then travels up the plate by capillary action, which is a balance between mobile phase surface tension and a retarding force such as viscosity or gravity. Additional separating power can be achieved if the plate is developed in two dimensions.
(2) Descending/radial technique – the stationary phase is not equilibrated with the mobile phase during the entire development process because the plate is dry until the solvent passes over it.
c. High–performance thin layer chromatography (HPTLC) – uses smaller stationary phase particles, thinner layer and controlled flow rates.
2. Column chromatography – a tube or column id fitted with particles of the stationary phase and the mobile phase is passed through the resultant chromatographic bed.
a. Gas chromatography – is a form of chromatography in which volatile solutes are separated by passing them through a column of stationary phase using an inert gas (e.g. nitrogen, helium or argon) as the mobile phase. Because the gaseous mobile phase “carries” the solute molecules through the column, it is often referred to as the carrier gas.
(1) Gas–solid chromatography – the stationary phase is particles of sorbent that have large surface area.
(2) Gas–liquid chromatography – a non–volatile liquid is coated onto particles of column packing or directly on the wall of chromatographic column.
Instrumentation in gas chromatography:
(1) Chromatographic columns – to separate the analytes being measured.
a. Packed columns – filled with particles that are used uncoated of the have been coated with stationary phase. Measures 0.1–0.4 cm in diameter and from 1–4 m or greater in length.
b. Capillary columns – the inner wall is coated with stationary phase. Measures 0.2 – 0.5 mm in diameter (I.D.) and from 10 – 150 m in length.
(2) Gas reservoir and flow control apparatus – to force the carrier gas through the injector, column and detector.
(3) Injector – for introducing an aliquot of sample or derivative analyte or analytes into the column.
(4) Column oven – to heat the column.
(5) Online detector – detects the separated analytes as they elute from the column.
a. Thermal conductivity detector (TCD) – is based on the principle that addition of a compound to a glass alters the thermal conductance of the gas. It is consist of a filament that is heated to a stable temperature in the presence of the carrier gas flow.
Other important operating parameters of the TCD include the detector temperature the flow rates of the carrier, make–up and reference gases and the corrosive potential of the compound to be analyzed. The detection of microbial metabolic patterns for identification of anaerobic microbes is one of the major applications of this detector in the clinical laboratory.
b. Flame ionization detector (FID) – the column effluent is mixed with hydrogen and air and the eluting compounds are burned by a flame.
c. Photoionization detector (PID) – the energy for ionization is provided by an intense ultraviolet lamp rather than by a flame.
d. Thermoionic Selective Detector (TSD) / Nitrogen–Phosphorous detector (NPD) – alkali bead selectively ionizes nitrogen or phosphorous containing compounds.
e. Electron capture detector (ECD) – based on the reaction between electromagnetic compounds and thermal electrons. The electrons are normally provided from a radioactive source such as nickel or tritium housed in the detector. A collector electrode is pulsed to collect “excess” electrons; this is called the standing current.
Gas chromatography/Mass spectrometry (GC/MS)
GC/MS is a powerful analytical technique that combines the resolving power of the gas chromatograph with the exquisite specificity and sensitivity of the mass spectrometer. GC/MS is used primarily for the analysis of drug.
Mass spectrometry (MS)
MS is a technique that involves the fragmentation of target molecule followed by the separation and measurement of the individual mass fragments. Consequently, it is an extremely useful instrument for determining the elemental composition and structure of both inorganic and organic compounds.
Because all compounds have mass, a mass spectrometer is considered a “universal” detector. MS is also capable of measuring specific mass fragments; thus it can be used to detect specific compounds even in a complex mixture.
AUTOMATION
When used in an industrial context, the term automation implies mechanical or electronic control of a process. In clinical chemistry, the same term is applied to the performance of analytical tests by an instrument or combination of components with only minor involvement of an analyst. Partial automation refers to procedures in which the initial preparation of a specimen is done manually, but in which the analysis precedes without human intervention.
Several steps are common to most analyses. These can be summarized as follows:
1. Sample pick up.
2. Sample delivery with or without subsequent washout of the sample probe with reagent or diluent.
3. Protein separation by precipitation, filtration, centrifugation, chromatographic technique or dialysis
4. The addition of one or more reagents, mixing and incubation.
5. Reaction detection by visible, UV or flame photometry, fluorometry or nephelometry.
6. Data presentation on a digital readout, strip chart recorder, printed tape or computer terminal
Factors contributing to advances in automation:
Developments in automation in the clinical laboratory are intertwined with advances in technology in a variety of fields. Three major areas contribute greatly to our ability to automate laboratory analysis and data handling:
1. Robotics
2. Computers and microprocessors
3. Sensors for monitoring reactions
As progress in any of these areas is made, the improvements are quickly incorporated into automated clinical analyzers.
Types of automatic analyzers:
1. Continuous flow analysis: segmented stream
Perhaps the earliest approach to automated chemical analysis was the continuous–flow system developed in the late 1950’s by Leonard Skeggs. The term continuous–flow describes the way in which the chemical and samples travel through the instrument. A unique characteristic of this system of analysis was the use of air bubbles in the sample and reagent streams. Air is injected into each stream as a series of small bubbles which travel along with the reaction system. The air bubbles minimize diffusion of reagents and mixing between samples, preserving the integrity of each individual reaction.
2. Continuous flow analysis: flow injection
Within 20 years of the inception of continuous–flow analysis, a radically different concept was developed. Instead of a stream segmented by air bubbles to promote sample integrity, direct injection of sample into very small diameter tubing was utilized for reactions and measurements. This new technique came to be called flow injection analysis.
In flow–injection analysis, the reagent stream is pumped through small tubing (usually about 0.5 mm in diameter). The sample slug is introduced directly into that stream, allowing intimate mixing within a few seconds.
3. Centrifugal analysis
The centrifugal analyzer was born in the mid–1960 as a result of discussions on alternative methods for automation. Although continuous–flow analysis had shown the value of automation, there was some satisfaction with its flexibility and ease of operation. Research at the OAK Ridge National Laboratory, directed by Dr. Norman Anderson, resulted in the development of an innovative approach to automated analysis called the centrifugal analyzer.
The components of the autoanalyzers are:
a. Sampler – a device that holds the cups containing the standards and specimens for analysis. The specimen cups are placed in a circular tray mounted on a spindle that rotates so that each specimen is presented in turn for analysis at a predetermined time. Aspiration of the sample is initiated by a programming cam which is selected to determine the rate of analysis, the dwell time in the specimen and the sample to wash ratio.
b. The pumps and manifolds – the pumps function on the principle of proportional addition of reagents and this depends on uniform delivery of solutions. The delivery of the samples, reagents and air into the analytical system depends on the diameter of the tubing. The total assembly of tubing and fittings is generally referred to as the manifold.
c. Dialyzers – dialysis is a process by which sample constituents of low molecular mass are separated from compounds with high molecular mass with the aid of a semi–permeable membrane. The diluted sample stream (donor stream) moves on one side of the membrane, while the recipient stream (reagent or saline) circulates through the other channel.
d. Heating baths are used to provide the elevated temperature and time delay required for the development of a colored reaction product or for utilization of substrate by an enzyme. For the basic autoanalyzer, a 40 foot glass coil immersed in mineral oil is used most often as a heating bath. The temperature is maintained around 37oC or 95oC in certain models, the set point can be altered by means of an adjustable thermoregulator.
e. Measurement or detection devices
(1) Colorimeter – employ a dual beam principle to reduce the effect of variation in the output of the light source due to voltage fluctuation.
(2) Flame photometers
(3) Fluorometer
(4) Spectrophotometer may be interfaced with an Autoanalyzer manifold to measure substances that absorb in the ultraviolet region.
(5) Atomic absorption flame photometers may be used for calcium, magnesium or trace elements analysis.
f. Data presentation
The autoanalyzer recorder, although physically discrete from colorimeter is in effect an integral part of the measuring system. In the early models, the recorder provided the only means of display data. The strip chart tracing indicates continuously the difference in voltage between test and reference photocells in the colorimeter.
A digital printer can be incorporated so that raw data from the linear output photometer may be converted directly into concentration units. This involves an analog to digital voltage converter.
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