Introduction
Think of your old hand-operated Spectronic 20, or your direct reading spectrophotometer that you use in your lab. You line up your samples in a row. In front of them, you place some small sample cups or maybe even a series of cuvettes, and you pipette a known estimate of sample into each cup. You then add a reagent and somehow mix the reagent and sample. You do this for each sample. You may have more reagents to add so you repeat the whole process until all reagents are added. Then you start a timer. When the timer beeps you know you have a positive "time window" to read the absorbance (or concentration) of your samples. You read by manually transferring the color-developed sample to a spectrometer cuvette, by using a peristaltic pump to replacement the sample to a flow cell already in the spectrometer, or by inserting the tube or cuvette that you used to form the sample color in. Then, you press a button to send the reading to a printer, a computer program, or you manually report the reading onto a laboratory worksheet.
Disposable Mouse Traps
Did you shake and mix every sample exactly the same way every time? Will you mix them the same way every day? Will every interpreter run them exactly the same way you have?
discrete Analyzers in the Environmental Laboratory
Is there color or turbidity in the samples? Should you zero your instrument with each sample, or only with reagent water blanks?
Is the exact time you read the final absorbance critical?
The process described is what you are automating by using a various analyzer. Instead of lining up samples, you are pouring aliquots into sample cups that are located on an auto sampler tray. Instead of transferring a known estimate of sample to a cuvette, the various analyzer does. Instead of adding reagents and mixing, the various analyzer does. Instead of starting a timer, the various analyzer does. Instead of reading the absorbance, recording the reading, and calculating a ensue the various analyzer does.
The analyzer has automatic approximately all the simple colorimetric methods for you. Sample volume is measured and dispensed exactly the same way, every time. Reagents are added and mixed exactly the same way every time. The timer is set and absorbance is measured exactly the same way every time. Results are calculated exactly the same way every time.
The various analyzer pipettes, dilutes, adds reagents, mixes, calibrates, measures, calculates, and reports all for you. You elect a recipe by keyboard. There is no hardware to manually change, no cartridge to rinse out, no baselines to monitor, no wavelength filters to change. Sample and reagent volumes are thought about by a option in a computer program, not by the internal diameter of a peristaltic pump tube.
The various analyzer has done a lot for you but it cannot operate nor do everything. It cannot accurately prepare the stock calibration suitable for you, even though it can accurately dilute it. It cannot certify the standards and samples were located on the auto sampler tray in the right order. It cannot prepare the reagents for you or certify they were located in the right order; however, it can monitor their Syn. Clearness and remind you where they are supposed to go. It cannot make sure you've entered the proper sample Id for each sample position, however, it can certify that the ensue obtained for that sample position is traceable to the Id you entered. It cannot know the sample lot Id for each suitable or reagent, but if you enter those Id's into the software, it can certify traceability of those reagents with your sample sets.
The software and built in electronics enduringly monitor and adjust lamp voltage so that absorbance readings do not drift. Drift is base in flow analyzers because the peristaltic pump tubing delivers reagents by proportion. The various analyzer delivers the exact estimate of sample and reagent every time. These volumes do not change. The various analyzer has a fixed path length if the various analyzer does not replacement color-developed sample to other cuvette, or flow cell, for measurement. In addition, if, the various analyzer reads through the walls of the cuvette the calibration curve is normally more stable and or reproducible than your reagents and standards.
Change your thoughts on calibration
Beer's law states that the absorbance is equal to the absorbtivity times the path length times the concentration. It seems, however, sometimes we do not believe that Beer's law is a law. I say this because according to this law, the absorbtivity is a constant. When the path length is fixed (always the same), the path length is a constant as well production the only variable the concentration. Therefore, you prepare standards of a known concentration, measure the absorbance and settle the absorbtivity. Assuming you can prepare reagents exactly the same way every time, measure the same volume every time, and incubate your samples the same estimate of time every time, there should be no fancy to assume that the absorbtivity would change. If the absorbtivity does not change, then there is no fancy to calibrate every day. Moreover, if the absorbtivity is not changing, you could admittedly be introducing error every time you calibrate because you may not be taking into catalogue random errors that occur in the middle of analysts or even with yourself as you inadvertently vary your technique on a day-to-day basis.
As mentioned previously, daily calibration is required for continuous flow methods because flow methods proportion the reagents and sample using a peristaltic pump. Those pump tubes are changing with time changing the relative proportion of sample and reagents. Flow analyzers are still incredibly accurate, it is just you need to calibrate each time.
Calibrating consumes time. Especially definite ones where you took great care to ensure your standards and reagents are fresh.
A hand-operated spectrometer does not necessarily want a calibration each time. Many methods written for hand-operated spectrometers merely say, "analyze a check suitable with each sample set". In fact, the stability of the calibration curve is the basal view behind direct reading spectrophotometers and filter wheel methods. For many colorimetric tests, the stability of the curve far exceeds the stability of the standards or the reagents. Some examples are nitrite and phosphate.
A various analyzer should not want daily calibrations and should allow us to extrapolate more the ion chromatography, gas chromatography, and hand-operated direct reading spectrometer view of the chronic Calibration Verification, or Ccv. As mentioned, the fancy the various analyzer curves are stable is that the robot exactly reproduces all every time. You cannot do this because you are not a robot, the various analyzer, however, is.
A hand-operated recipe uses more reagent and sample volume because we, as humans, cannot work admittedly with small volumes. A flow theory uses more reagent than a various analyzer because a flow instrument is continuously pumping reagent through the system.
Discrete analyzers that measure the sample absorbance within the same box that the reaction occurred originate less waste than instruments that wash the vessel, or use a flow cell. In fact, adequately rinsing a flow cell requires necessary rinsing in the middle of samples production the waste volume generated essentially equivalent to that of a micro-flow Segmented Flow Analyzer, or Low Flow Flow Injection Analyzer.
The various analyzer uses significantly less reagent, and generates significantly less waste than hand-operated methods. This chart illustrates an unscaled down hand-operated recipe using the exact volumes described in suitable Methods. The waste generated for the hand-operated recipe does not take into catalogue washing of glassware. As mentioned earlier, an analyzer that washes cuvettes or rinses a flow cell will originate more waste than indicated here.
Eliminate the possibility of contamination, or false positives
The various analyzer measuring the absorbance of a color reacted sample contained in private cuvettes. Unlike flow analysis, there is no possibility of interaction in the middle of samples and unlike flow analysis; the user can visually contemplate the reaction stock while and after analysis.
Using a various analyzer, the interpreter can contemplate the reaction while color development and after the test is complete. The interpreter can remove the reaction segments and verify that dispensed volumes are repeatable, that there are no bubbles or turbidity, and that the color looks correct. A flow analyzer does not give the interpreter the capability to visually contemplate and qualitatively certify the accuracy of his or her results.
A various analyzer dispenses, reacts, incubates, and measures all within the reaction cuvette without transferring to a flow cell. Analyzers that replacement to a flow cell are not "true" various analyzers, but instead, are hybrids in the middle of flow and discrete. The hybridization is done to perform lower detection limits; however, the advantage of the individually contained reaction and absence of carryover is lost. In addition, since these analyzers want as much rinse as a flow analyzer to remove preceding samples, waste generation is as high as flow. Given this, and the increased possibility of environmental contamination or analyte loss that occurs from open-air heated reactions, you may as well have a flow analyzer.
Chemical reactions occur in individually contained segments
All various analyzers have reaction segments. Some analyzers do chemical reactions in a cuvette segment and then replacement the reacted sample to a flow cell. This type of analyzer is a hybrid of various and flow, and not a true various analyzer. A true various analyzer reacts and measures the sample within the optic cuvette. Some analyzers wash the optic cuvette in the middle of tests. Washing in the middle of tests enables more samples to be analyzed per cuvette; however, the washing cannot certify that there is no residual contamination that remaining after the washing process. Other various analyzers utilize disposable optic capability cuvettes.
Washing in the middle of tests enables more samples to be analyzed per cuvette; however, the washing cannot certify that there is no residual contamination not thoroughly removed by the washing process. This residual contamination can come from preceding samples, or more likely, from the reagents used in processing the preceding samples. The built in computerized checking of optic capability cannot verify absence of chemical contamination.
Analyzers that use a flow cell still react samples in some sort of cuvette. It is the estimate of reaction vessels on the various analyzer that limit the estimate of tests that the various can run in a singular walk away operation. If the various analyzer has 100 sample positions and 200 reaction cuvettes, then the analyzer can run 100 samples for 2 tests each. The various analyzer with the flow cell must rinse the flow cell in the middle of each sample, and rinse vigorously in the middle of each test. Consider that a two-channel flow analyzer can analyze 100 samples for two tests each in less than half the time as a various analyzer with a flow cell. Also, Consider that the flow analyzer generates no more waste than the various analyzer with a flow cell. If the required testing is a lot of samples for one or two tests it makes more sense to use a flow analyzer.
Reagents can interfere as cross contamination in the middle of samples. Using disposable private reaction cuvettes thoroughly eliminates the possibility of contamination. For instance, the cadmium reduction nitrate test contains necessary amounts of ammonia in the buffer reagent and phosphate in the color reagent. Using private disposable cuvettes ensures that there is no contamination. Washing cuvettes, or using a flow cell, means you can never be sure.
Using disposable optic cuvettes is the only way you can certify no carryover in the middle of tests or samples. The view is similar to use of disposable petri dishes, disposable pipette tips, and disposable hypodermic needles. The various analyzer admittedly and rapidly analyzes manifold tests on singular sample solutions. Only disposable individually contained reactions ensure that there is no interaction in the middle of samples or tests.
Let the robot do your pipetting.
When you manually pipette samples you, hopefully, use a separate pipette per sample. If not, you will at least rinse it in in the middle of samples, and possibly with sample prior to transferring your sample aliquot to the sample container. This is to avoid carryover in the middle of samples. A flow analyzer uses an auto sampler. The sampling probe immerses in the wash center rinsing the surface of the probe, and pulls wash clarification from the center and into the analytical cartridge.
A various analyzer also uses a probe; however, it operates differently than flow analyzers. A various analyzer's level detect mechanism ensures that the probe immerses into the sample or reagents no additional than necessary to withdraw the required sample aliquot. The probe then washes itself on the surface at the wash center and pushes the sample or reagent out into the sample cuvette. in the middle of dispenses, the probe pushes excess wash water out ensuring no carryover. In other words, unlike a flow theory that only pulls sample in one direction, the sampling probe on a various analyzer is bidirectional pulling reagent and sample into its internal tubing only far sufficient to withdraw the definite volume and then dispensing it by pushing it out the other way.
The machine can think.
When doing a hand-operated test you know if you ran out of reagent or sample. A flow analyzer does not know. A flow analyzer could end up aspirating from empty sample cups or empty reagent bottles all night long and think it is still running samples. A various analyzer with level detection prevents this. The level detect mechanism is a capacitance detector that senses the distinction in the middle of liquid and air. The various software calculates the volume of reagents and samples based on the height of liquid. The software continuously monitors sample and reagent volumes and will not continue the test when it detects that reagents or samples have "run out".
The sampling depth on a flow analyzer is normally adjustable by the user and is normally towards the lowest of the sample vial. On a various analyzer, the depth the probe immerses in a sample clarification is a ensue of programming or instrument design. The depth sampled on the Oi various analyzer is thought about by the level detect mechanism and the sample aliquot required for the test. For instance, if 200 micro liters is required the probe will immerse just below 200 micro liters as thought about by the volume of the cup and the liquid level detected and withdraw a software-defined estimate above 200 micro liters. In other words, the various analyzer samples from the top 300 micro liters of sample solution. The probe only immerses as far as it has to. This minimizes inherent carryover contamination, and speeds the process. In this way dispensing and rinsing is fast and there is no sample or reagent carried to other on the sides of the probe.
When sampling from the top of the sample cup there is a risk of loss of a vaporing analyte from the top of the clarification or the risk of the adsorption of an analyte from the laboratory air into the top of the solution. For instance, trace cyanide in near neutral clarification can be slowly lost from the top layer of sample clarification into the lab air. This is especially clear with lower concentrations such as 10 ppb.
Gain of the analyte is inherent as well. Ammonia is a base laboratory contaminant. Ammonia easily adsorbs into acidified solutions. It is inherent for ammonia to be "pulled" from laboratory air into the sample solution. A flow analyzer would not as easily detect this loss or gain because it samples from the lowest of the sample cup.
There are some drawbacks
A various analyzer reacts sample in a heated cup that is open to allow the probe to dispense samples and reagents. The heat increases reaction rates and is especially important for chemistries such as ammonia that are slow to form color. In hand-operated testing the reagents are added in open containers, however, the box shape can vary and the box can be capped while mixing, heating, and color reaction. When flow analyzers were first introduced one of the key advantages that gained its acceptance over hand-operated methods was that reactions occurred enclosed within the tubing limiting its exposure to laboratory air. In this aspect, various analyzers are kind of a step backwards.
There are necessary advantages.
Similar to keeping a color developing reaction in its own box till it reaches a color maximum, various analyzers can also hold intermediate reactions for long periods of time without risk of carryover, dilution into a carrier reagents, or excessive dispersion. This can be especially beneficial in enzyme or reduction reactions where reaction rates are slow. A flow analyzer would want long delay coils resulting in very complicated Sfa chemistry manifolds. Often elevated temperature is used to speed reactions, but in some chemistry, there are limits to the maximum temperatures possible. Since various analyzer reactions are occurring in individually contained cuvettes, the time delay in the middle of reagent additions on various analyzers is minute only by software. This is a necessary advantage over flow chemistry.
In hand-operated methods, obviously, the operator prepares all the calibration standards from a stock solution, dilutes any Qc samples from a stock solution, dilutes samples known to be over calibration prior to color development, and dilutes samples that were over calibration once he or she notices that they are. Unless you have an added auto-dilutor attached to your flow analyzer, you will still be diluting standards and over calibration samples. Auto-dilution is an integral function of a various analyzer. The dilutions can be preset while sample table entry if you know that the samples need to be diluted. Methods can be programmed such that they dilute every sample and suitable all the time, or the instrument can be programmed so that over calibration, samples are diluted and re analyzed.
An interpreter changes a hand-operated or flow recipe from one to the next by memory, or by referring to the Sop. How well this singular interpreter performs the course is dependent upon his mood, the time of day, his feel with the method, the availability of equipment, and many other unquantifiable variables. It is inherent to procure good results and bad results by the same manually performed method. A flow analyzer analyzes all the same every time assuming it is set up the same every time. This assumption is valid with experienced flow analysis technicians; however, if the technician does not understand flow or if there are manifold users results will vary. Extensive training and documentation is necessary to certify that results conform to good automatic lab practices.
The various analyzer recipe is prime by mouse click when scheduling analyses on the sample tray. The recipe conditions do not change. In fact, assuming you have accurately calibrated your recipe the calibration is stored within the method. This means that an untrained interpreter that only knows what buttons to press is able to procure same results to even the most experienced analyst.
Most analytes performed in an environmental compliance laboratory cannot be bench spiked. If the analyte requires a introductory distillation, digestion, or dismissal the spiking is done prior to the introductory sample process. I perceive that many labs do not distill ammonia or Fluoride and I would argue that if you are reporting compliance testing for the clean water act you would good seriously Consider changing your Sop. Other parameters that can't be spiked are those that are too high to spike within the matrix without introductory dilution, such as Ca, Mg, Cl, So4, and analytes like alkalinity that just are not spiked.
This shortens the list of inherent analytes for the automatic spiking function to nitrite, phosphate, Sulfide, Chromium Vi, and some others. On these, I defer back to the old slide and ask if the inherent error is worth the risk for so few tests.
Summary
Benefits of various analyzers comprise decreased reagent consumption, decreased waste generated, and ease of use among other things. The most necessary advantage of the various analyzer, however, is that it can eliminate the original view of disposition analysis and allow you to run samples as you receive them instead of storing them until there is sufficient sitting around to make a flow or Ic analysis worthwhile. If you take advantage of the calibration stability of the various analyzer, and accurately prepare a calibration that can then be used by approximately any interpreter in subsequent uses an added advantage is that the results are the same regardless of who uses the machine.
Think of those short keeping time samples. The phosphate, the nitrites, the chromium Vi, and residual chlorine. These analytes cause the environmental lab to stop all just to get the analysis done on time. Think of the other analytes that come in periodically, but maybe not frequently. possibly silica, ferrous iron and sulfide. How do you certify these tests followed the Sop? Instead of mental of the various analyzer as something to replace a flow instrument, think of it as something to supplement a flow instrument. If you have hundreds of samples for one or two tests routinely and for the same analyte you are not going to save money by switching these tests to a various analyzer. Where you will save money and great effort is removing unnecessary strain from the flow analyzer and your analysts by performing the non - disposition or "rush" tests on a various analyzer. It is inherent for the sample login someone to analyze samples as received for approximately every colorimetric test that does not want a digestion. In other words, as soon as the sample is logged in it could be immediately run for nitrite, phosphate, chromium Vi, nitrate, ammonia, chloride, and sulfate. In this example, instead of putting samples in a refrigerator to be gathered for analysis at a later time, they end up being run by ice chest and by client as soon as they are received.
If all is to run on the various analyzer, then procure your samples in a vial that fits on the various analyzer. You no longer need to replacement liquid from box A to auto sampler vial B, the sample bottle can be the auto sampler vial. Not only does this save time, but it saves shipping as well. Instead of large ice chests, you use tiny mailers.
To summarize, the true advantage of a various analyzer is that its built in features allow any interpreter to get the same results every time. various analyzers are very simple to use requiring minimal software training. Once set up for your laboratory, properly applied methods allow you to modify your daily routines and analyze samples as soon as they come in. Whether you are an environmental lab, research, process control, or municipality various analyzers can be used effectively in your operation. Currently, the full power of various analyzers is minute by tradition and by regulation. Once we start to form methods for various analyzers instead of using various analyzers to run methods industrialized for flow we will be able to see greater throughput, less variability, and lower Mdl.
discrete Analyzers in the Environmental Laboratory
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