The AutoAnalyzer is an automated analyzer using a flow technique called continuous flow analysis (CFA), or more correctly segmented flow analysis (SFA) first made by the Technicon Corporation. The instrument was invented in 1957 by Leonard Skeggs, PhD and commercialized by Jack Whitehead's Technicon Corporation. The first applications were for clinical analysis, but methods for industrial and environmental analysis soon followed. The design is based on segmenting a continuously flowing stream with air bubbles.
Continuous flow analysis (CFA) is a general term that encompasses both segmented flow analysis (SFA) and flow injection analysis (FIA). In segmented flow analysis, a continuous stream of material is divided by air bubbles into discrete segments in which chemical reactions occur. The continuous stream of liquid samples and reagents are combined and transported in tubing and mixing coils. The tubing passes the samples from one apparatus to the other with each apparatus performing different functions, such as distillation, dialysis, extraction, ion exchange, heating, incubation, and subsequent recording of a signal. An essential principle of SFA is the introduction of air bubbles. The air bubbles segment each sample into discrete packets and act as a barrier between packets to prevent cross contamination as they travel down the length of the glass tubing. The air bubbles also assist mixing by creating turbulent flow (bolus flow), and provide operators with a quick and easy check of the flow characteristics of the liquid. Samples and standards are treated in an exactly identical manner as they travel the length of the fluidic pathway, eliminating the necessity of a steady state signal, however, since the presence of bubbles create an almost square wave profile, bringing the system to steady state does not significantly decrease throughput ( third generation CFA analyzers average 90 or more samples per hour) and is desirable in that steady state signals (chemical equilibrium) are more accurate and reproducible.[1] Reaching steady state enables lowest detection limits to be reached.
A continuous segmented flow analyzer (SFA) consists of different modules including a sampler, pump, mixing coils, optional sample treatments (dialysis, distillation, heating, etc.), a detector, and data generator. Most continuous flow analyzers depend on color reactions using a flow through photometer, however, also methods have been developed that use ISE, flame photometry, ICAP, fluorometry, and so forth.
Flow injection analysis (FIA), was introduced in 1975 by Ruzicka and Hansen,[2] The first generation of FIA technology, termed flow injection (FI), was inspired by the AutoAnalyzer technique invented by Skeggs in early 1950s.[3][4] While Skeggs' AutoAnalyzer uses air segmentation to separate a flowing stream into numerous discrete segments to establish a long train of individual samples moving through a flow channel, FIA systems separate each sample from subsequent sample with a carrier reagent. While the AutoAnalyzer mixes sample homogeneously with reagents, in all FIA techniques sample and reagents are merged to form a concentration gradient that yields analysis results.
FIA methods can be used for both fast reactions as well as slow reactions. For slow reactions, a heater is often utilized. The reaction does not need to reach completion since all samples and standards are given the same period to react. For typical assays commonly measured with FIA (e.g., nitrite, nitrate, ammonia, phosphate) it is not uncommon to have a throughput of 60-120 samples per hour.
FIA methods are limited by the amount of time necessary to obtain a measurable signal since travel time through the tubing tends to broaden peaks to the point where samples can merge with each other. As a general rule, FIA methods should not be used if an adequate signal cannot be obtained within two minutes, and preferably less than one.[citation needed] Reactions that need longer reaction times should be segmented. However, considering the number of FIA publications and wide variety of uses of FIA for serial assays, the "one minute" time limitation does not seem to be a serious limitation for most real life assays.[citation needed] Yet, assays based on slow chemical reactions have to be carried either in stopped flow mode ( SIA) or by segmenting the flow.
OI Analytical, in its gas diffusion amperometric total cyanide method, uses a segmented flow injection analysis technique that allows reaction times of up to 10 minutes by flow injection analysis.[5]
Technicon experimented with FIA long before it was championed by Ruzicka and Hansen. Andres Ferrari reported that analysis was possible without bubbles if flow rates were increased and tubing diameters decreased.[6] In fact, Skegg's first attempts at the auto analyzer did not segment. Technicon chose to not pursue FIA because it increased reagent consumption and the cost of analysis.[citation needed]
The second generation of the FIA technique, called sequential injection analysis (SIA), was conceived in 1990 by Ruzicka and Marshal, and has been further developed and miniaturized over the course of the following decade.[citation needed] It uses flow programming instead of the continuous flow regime (as used by CFA and FIA), that allows the flow rate and flow direction to be tailored to the need of individual steps of analytical protocol. Reactants are mixed by flow reversals and a measurement is carried out while the reaction mixture is arrested within the detector by stopping the flow. Microminiaturized chromatography is carried out on microcolumns that are automatically renewed by microfluidic manipulations. The discrete pumping and metering of microliter sample and reagent volumes used in SI only generates waste per each sample injection. The enormous volume of FI and SI literature documents the versatility of FI and SI and their usefulness for routine assays (in soil, water, environmental, biochemical and biotechnological assays) has demonstrated their potential to be used as a versatile research tool.
Dialyzer module
In medical testing applications and industrial samples with high concentrations or interfering material, there is often a dialyzer module in the instrument in which the analyte permeates through a dialysis membrane into a separate flow path going on to further analysis. The purpose of a dialyzer is to separate the analyte from interfering substances such as protein, whose large molecules do not go through the dialysis membrane but go to a separate waste stream. The reagents, sample and reagent volumes, flow rates, and other aspects of the instrument analysis depend on which analyte is being measured. The autoanalyzer is also a very small machine
Recording of results
Previously a chart recorder and more recently a data logger or personal computer records the detector output as a function of time so that each sample output appears as a peak whose height depends on the analyte level in the sample.
Commercialization
The AutoAnalyzer setup (1966)
Technicon sold its business to Revlon in 1980 [7] who later sold the company to separate clinical (Bayer) and industrial (Bran+Luebbe - now SEAL Analytical) buyers in 1987. At the time, industrial applications accounted for about 20% of CFA machines sold.
In 1974 Ruzicka and Hansen carried out in Denmark and in Brasil initial experiments on a competitive technique, that they termed flow injection analysis (FIA). Since then the technique found worldwide use in research and routine applications, and was further modified through miniaturization and by replacing continuous flow with computer controlled programmable flow.
During the 1960s industrial laboratories were hesitant to use the autoanalyzer. Acceptance by regulatory agencies eventually came about by demonstration that the techniques are no different from a recording spectrophotometer with reagents and samples added at the exact chemical ratios as traditionally accepted manual methods.[8]
The best known of Technicon's CFA instruments are the AutoAnalyzer II (introduced 1970), the Sequential Multiple Analyzer (SMA, 1969), and the Sequential Multiple Analyzer with Computer (SMAC, 1974). The Autoanalyzer II (AAII) is the instrument that most EPA methods were written on and reference.[citation needed] The AAII is a second generation segmented flow analyzer that uses 2 millimeter ID glass tubing and pumps reagent at flow rates of 2 - 3 milliliters per minute. Typical sample throughput for the AAII is 30 - 60 samples per hour.[9] Third generation segmented flow analyzers were proposed in the literature,[10] but not developed commercially until Alpkem introduced the RFA 300 in 1984. The RFA 300 pumps at flow rates less than 1 milliliter per minute through 1 millimeter ID glass mixing coils. Throughput on the RFA can approach 360 samples per hour, but averages closer to 90 samples per hour on most environmental tests. In 1986, Technicon (Bran+Luebbe) introduced its own microflow TRAACS-800 system.[11]
Bran+Luebbe continued to manufacture the AutoAnalyzer II and TRAACS, a micro-flow analyzer for environmental and other samples, introduced the AutoAnalyzer 3 in 1997 and the QuAAtro in 2004. The Bran+Luebbe CFA business was bought by SEAL Analytical in 2006 and they continue to manufacture, sell and support the AutoAnalyzer II/3 and QuAAtro CFA systems, as well as Discrete Analyzers.
And there are other manufacturers of CFA instruments.
Skalar Inc., subsidiary of Skalar Analytical, founded in 1965, which has its head office in Breda (NL), is since its founding an independent company, fully owned by its personnel. Development in robotic analyzers, TOC and TN equipment, and monitors has extended the product lines of its long life SAN++ Continuous Flow Analyzers. Software packages for data acquisition and analyzer control are also in house products, running with latest software demands and handles all analyzer hardware combinations.
Astoria-Pacific International, for example, was founded in 1990 by Raymond Pavitt, who previously owned Alpkem. Based in Clackamas, Oregon, U.S.A., Astoria-Pacific manufactures its own micro-flow systems. Its products include the Astoria Analyzer lines for Environmental and Industrial applications; the SPOTCHECK Analyzer for Neonatal screening; and FASPac (Flow Analysis Software Package) for data acquisition and computer interface.
FIAlab Instruments, Inc., in Seattle, Washington, also manufactures several analyzer systems.
Alpkem was purchased by Perstorp Group, and then later by OI Analytical in College Station Texas. OI Analytical manufactures the only segmented flow analyzer that uses polymeric tubing in place of glass mixing coils. OI is also the only major instrument manufacturer that provides segmented flow analysis (SFA) and flow injection analysis (FIA) options on the same platform.
Clinical analysis
AutoAnalyzers were used mainly for routine repetitive medical laboratoryanalyses, but they had been replaced during the last years more and more by discrete working systems which allow lower reagent consumption. These instruments typically determine levels of albumin, alkaline phosphatase, aspartate transaminase (AST), blood urea nitrogen, bilirubin, calcium, cholesterol, creatinine, glucose, inorganic phosphorus, proteins, and uric acid in blood serum or other bodily samples. AutoAnalyzers automate repetitive sample analysis steps which would otherwise be done manually by a technician, for such medical tests as the ones mentioned previously. This way, an AutoAnalyzer can analyze hundreds of samples every day with one operating technician. Early AutoAnalyzer instruments each tested multiple samples sequentially for individual analytes. Later model AutoAnalyzers such as the SMAC tested for multiple analytes simultaneously in the samples.
In 1959 a competitive system of analysis was introduced by Hans Baruch of Research Specialties Company. That system became known as Discrete Sample Analysis and was represented by an instrument known as the "Robot Chemist." Over the years the Discrete Sample Analysis method slowly replaced the Continuous Flow system in the clinical laboratory.[12]
Industrial analysis
The first industrial applications - mainly for water, soil extracts and fertilizer - used the same hardware and techniques as clinical methods, but from the mid-1970s special techniques and modules were developed so that by 1990 it was possible to perform solvent extraction, distillation, on-line filtration and UV digestion in the continuously flowing stream. In 2005 about two thirds of systems sold worldwide were for water analysis of all kinds,[citation needed] ranging from sub-ppb levels of nutrients in seawater to much higher levels in waste water; other common applications are for soil, plant, tobacco, food, fertilizer and wine analysis.
Current Uses
AutoAnalyzers are still used for a few clinical applications such as neonatal screening or Anti-D, but the majority of instruments are now used for industrial and environmental work. Standardized methods have been published by the ASTM (ASTM International), the US Environmental Protection Agency (EPA) as well as the International Organization for Standardization (ISO) for environmental analytes such as nitrite, nitrate, ammonia, cyanide, and phenol. Autoanalyzers are also commonly used in soil testing laboratories, fertilizer analysis, process control, seawater analysis, air contaminants, and tobacco leaf analysis.
Technicon published method sheets for a wide range of analyses and a few of these are listed below. These methods and later methods are available from SEAL Analytical. Method lists for manufacturers instruments are readily available on their websites.
Sheet no.
Determination
Sample
Main reagent(s)
Colorimeter
N-1c
Urea nitrogen
Blood or urine
Diacetyl monoxime
520nm
N-2b
Glucose
Blood
Potassium ferricyanide
420nm
N-3b
Kjeldahl nitrogen
Foodstuffs
Phenol & hypochlorite
630nm
P-3b
Phosphate
Boiler water
Molybdate
650nm
Notes
↑ Coakly, William A., Handbook of Automated Analysis, Mercel Dekker, 1981 p 61
↑ J., Rulika; Hansen, E. H. (1975). "Flow injection analyses: I. New concept of fast continuous-flow analysis". Anal. Chim. Acta. 78: 145–157. doi:10.1016/S0003-2670(01)84761-9.
Analytical chemistry studies and uses instruments and methods to separate, identify, and quantify matter. In practice, separation, identification or quantification may constitute the entire analysis or be combined with another method. Separation isolates analytes. Qualitative analysis identifies analytes, while quantitative analysis determines the numerical amount or concentration.
Titration is a common laboratory method of quantitative chemical analysis to determine the concentration of an identified analyte. A reagent, termed the titrant or titrator, is prepared as a standard solution of known concentration and volume. The titrant reacts with a solution of analyte to determine the analyte's concentration. The volume of titrant that reacted with the analyte is termed the titration volume.
Inductively coupled plasma mass spectrometry (ICP-MS) is a type of mass spectrometry that uses an inductively coupled plasma to ionize the sample. It atomizes the sample and creates atomic and small polyatomic ions, which are then detected. It is known and used for its ability to detect metals and several non-metals in liquid samples at very low concentrations. It can detect different isotopes of the same element, which makes it a versatile tool in isotopic labeling.
The enzyme-linked immunosorbent assay (ELISA) is a commonly used analytical biochemistry assay, first described by Eva Engvall and Peter Perlmann in 1971. The assay is a solid-phase type of enzyme immunoassay (EIA) to detect the presence of a ligand in a liquid sample using antibodies directed against the protein to be measured. ELISA has been used as a diagnostic tool in medicine, plant pathology, and biotechnology, as well as a quality control check in various industries.
An automated analyser is a medical laboratory instrument designed to measure various substances and other characteristics in a number of biological samples quickly, with minimal human assistance. These measured properties of blood and other fluids may be useful in the diagnosis of disease.
An assay is an investigative (analytic) procedure in laboratory medicine, mining, pharmacology, environmental biology and molecular biology for qualitatively assessing or quantitatively measuring the presence, amount, or functional activity of a target entity. The measured entity is often called the analyte, the measurand, or the target of the assay. The analyte can be a drug, biochemical substance, chemical element or compound, or cell in an organism or organic sample. An assay usually aims to measure an analyte's intensive property and express it in the relevant measurement unit.
In analytical chemistry, a calibration curve, also known as a standard curve, is a general method for determining the concentration of a substance in an unknown sample by comparing the unknown to a set of standard samples of known concentration. A calibration curve is one approach to the problem of instrument calibration; other standard approaches may mix the standard into the unknown, giving an internal standard. The calibration curve is a plot of how the instrumental response, the so-called analytical signal, changes with the concentration of the analyte.
Gas chromatography (GC) is a common type of chromatography used in analytical chemistry for separating and analyzing compounds that can be vaporized without decomposition. Typical uses of GC include testing the purity of a particular substance, or separating the different components of a mixture. In preparative chromatography, GC can be used to prepare pure compounds from a mixture.
Capillary electrophoresis (CE) is a family of electrokinetic separation methods performed in submillimeter diameter capillaries and in micro- and nanofluidic channels. Very often, CE refers to capillary zone electrophoresis (CZE), but other electrophoretic techniques including capillary gel electrophoresis (CGE), capillary isoelectric focusing (CIEF), capillary isotachophoresis and micellar electrokinetic chromatography (MEKC) belong also to this class of methods. In CE methods, analytes migrate through electrolyte solutions under the influence of an electric field. Analytes can be separated according to ionic mobility and/or partitioning into an alternate phase via non-covalent interactions. Additionally, analytes may be concentrated or "focused" by means of gradients in conductivity and pH.
An immunoassay (IA) is a biochemical test that measures the presence or concentration of a macromolecule or a small molecule in a solution through the use of an antibody (usually) or an antigen (sometimes). The molecule detected by the immunoassay is often referred to as an "analyte" and is in many cases a protein, although it may be other kinds of molecules, of different sizes and types, as long as the proper antibodies that have the required properties for the assay are developed. Analytes in biological liquids such as serum or urine are frequently measured using immunoassays for medical and research purposes.
Total organic carbon (TOC) is an analytical parameter representing the concentration of organic carbon in a sample. TOC determinations are made in a variety of application areas. For example, TOC may be used as a non-specific indicator of water quality, or TOC of source rock may be used as one factor in evaluating a petroleum play. For marine surface sediments average TOC content is 0.5% in the deep ocean, and 2% along the eastern margins.
Liquid chromatography–mass spectrometry (LC–MS) is an analytical chemistry technique that combines the physical separation capabilities of liquid chromatography with the mass analysis capabilities of mass spectrometry (MS). Coupled chromatography – MS systems are popular in chemical analysis because the individual capabilities of each technique are enhanced synergistically. While liquid chromatography separates mixtures with multiple components, mass spectrometry provides spectral information that may help to identify each separated component. MS is not only sensitive, but provides selective detection, relieving the need for complete chromatographic separation. LC–MS is also appropriate for metabolomics because of its good coverage of a wide range of chemicals. This tandem technique can be used to analyze biochemical, organic, and inorganic compounds commonly found in complex samples of environmental and biological origin. Therefore, LC–MS may be applied in a wide range of sectors including biotechnology, environment monitoring, food processing, and pharmaceutical, agrochemical, and cosmetic industries. Since the early 2000s, LC–MS has also begun to be used in clinical applications.
An analyser or analyzer is a tool used to analyze data. For example, a gas analyzer tool is used to analyze gases. It examines the given data and tries to find patterns and relationships. An analyser can be a piece of hardware or software.
The carryover effect is a term used in clinical chemistry to describe the transfer of unwanted material from one container or mixture to another. It describes the influence of one sample upon the following one. It may be from a specimen, or a reagent, or even the washing medium. The significance of carry over is that even a small amount can lead to erroneous results.
Flow injection analysis (FIA) is an approach to chemical analysis. It is accomplished by injecting a plug of sample into a flowing carrier stream. The principle is similar to that of Segmented Flow Analysis (SFA) but no air is injected into the sample or reagent streams..
Proton-transfer-reaction mass spectrometry (PTR-MS) is an analytical chemistry technique that uses gas phase hydronium reagent ions which are produced in an ion source. PTR-MS is used for online monitoring of volatile organic compounds (VOCs) in ambient air and was developed in 1995 by scientists at the Institut für Ionenphysik at the Leopold-Franzens University in Innsbruck, Austria. A PTR-MS instrument consists of an ion source that is directly connected to a drift tube and an analyzing system. Commercially available PTR-MS instruments have a response time of about 100 ms and reach a detection limit in the single digit pptv or even ppqv region. Established fields of application are environmental research, food and flavor science, biological research, medicine, security, cleanroom monitoring, etc.
Capillary electrophoresis–mass spectrometry (CE–MS) is an analytical chemistry technique formed by the combination of the liquid separation process of capillary electrophoresis with mass spectrometry. CE–MS combines advantages of both CE and MS to provide high separation efficiency and molecular mass information in a single analysis. It has high resolving power and sensitivity, requires minimal volume and can analyze at high speed. Ions are typically formed by electrospray ionization, but they can also be formed by matrix-assisted laser desorption/ionization or other ionization techniques. It has applications in basic research in proteomics and quantitative analysis of biomolecules as well as in clinical medicine. Since its introduction in 1987, new developments and applications have made CE-MS a powerful separation and identification technique. Use of CE–MS has increased for protein and peptides analysis and other biomolecules. However, the development of online CE–MS is not without challenges. Understanding of CE, the interface setup, ionization technique and mass detection system is important to tackle problems while coupling capillary electrophoresis to mass spectrometry.
A triple quadrupole mass spectrometer (TQMS), is a tandem mass spectrometer consisting of two quadrupole mass analyzers in series, with a (non-mass-resolving) radio frequency (RF)–only quadrupole between them to act as a cell for collision-induced dissociation. This configuration is often abbreviated QqQ, here Q1q2Q3.
Mass spectrometric immunoassay (MSIA) is a rapid method is used to detect and/ or quantify antigens and or antibody analytes. This method uses an analyte affinity isolation to extract targeted molecules and internal standards from biological fluid in preparation for matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS). This method allows for "top down" and "bottom up" analysis. This sensitive method allows for a new and improved process for detecting multiple antigens and antibodies in a single assay. This assay is also capable of distinguishing mass shifted forms of the same molecule via a panantibody, as well as distinguish point mutations in proteins. Each specific form is detected uniquely based on their characteristic molecular mass. MSIA has dual specificity because of the antibody-antigen reaction coupled with the power of a mass spectrometer.
Droplet-based microfluidics manipulate discrete volumes of fluids in immiscible phases with low Reynolds number and laminar flow regimes. Interest in droplet-based microfluidics systems has been growing substantially in past decades. Microdroplets offer the feasibility of handling miniature volumes of fluids conveniently, provide better mixing, encapsulation, sorting, sensing and are suitable for high throughput experiments. Two immiscible phases used for the droplet based systems are referred to as the continuous phase and dispersed phase.
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