Think of your old manual 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 amount 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 certain “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 transfer the sample to a flow cell already in the spectrometer, or by inserting the tube or cuvette that you used to develop the sample color in. Then, you press a button to send the reading to a printer, a computer program, or you manually record the reading onto a laboratory worksheet.
Did you shake and mix every sample exactly the same way every time? Will you mix them the same way every day? Will every analyst run them exactly the same way you have?
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 discrete analyzer. Instead of lining up samples, you are pouring aliquots into sample cups that are placed on an auto sampler tray. Instead of transferring a known amount of sample to a cuvette, the discrete analyzer does. Instead of adding reagents and mixing, the discrete analyzer does. Instead of starting a timer, the discrete analyzer does. Instead of reading the absorbance, recording the reading, and calculating a result the discrete analyzer does.
The analyzer has automated almost 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 discrete analyzer pipettes, dilutes, adds reagents, mixes, calibrates, measures, calculates, and reports all for you. You select a method 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 determined by a selection in a computer program, not by the internal diameter of a peristaltic pump tube.
The discrete analyzer has done a lot for you but it cannot control nor do everything. It cannot accurately prepare the stock calibration standard for you, even though it can accurately dilute it. It cannot guarantee the standards and samples were placed on the auto sampler tray in the right order. It cannot prepare the reagents for you or guarantee they were placed in the right order; however, it can monitor their purity 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 guarantee that the result obtained for that sample position is traceable to the ID you entered. It cannot know the sample lot ID for each standard or reagent, but if you enter those ID’s into the software, it can guarantee traceability of those reagents with your sample sets.
The software and built in electronics constantly monitor and adjust lamp voltage so that absorbance readings do not drift. Drift is common in flow analyzers because the peristaltic pump tubing delivers reagents by proportion. The discrete analyzer delivers the exact amount of sample and reagent every time. These volumes do not change. The discrete analyzer has a fixed path length if the discrete analyzer does not transfer color-developed sample to another cuvette, or flow cell, for measurement. In addition, if, the discrete analyzer reads through the walls of the cuvette the calibration curve is usually 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 making the only variable the concentration. Therefore, you prepare standards of a known concentration, measure the absorbance and determine 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 amount of time every time, there should be no reason to assume that the absorbtivity would change. If the absorbtivity does not change, then there is no reason to calibrate every day. Moreover, if the absorbtivity is not changing, you could actually be introducing error every time you calibrate because you may not be taking into account random errors that occur between 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 accurate ones where you took great care to ensure your standards and reagents are fresh.
A manual spectrometer does not necessarily require a calibration each time. Many methods written for manual spectrometers merely say, “analyze a check standard with each sample set”. In fact, the stability of the calibration curve is the underlying concept 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 discrete analyzer should not require daily calibrations and should allow us to extrapolate more the ion chromatography, gas chromatography, and manual direct reading spectrometer concept of the Continuing Calibration Verification, or CCV. As mentioned, the reason the discrete analyzer curves are stable is that the robot exactly reproduces everything every time. You cannot do this because you are not a robot, the discrete analyzer, however, is.
A manual method uses more reagent and sample volume because we, as humans, cannot work easily with small volumes. A flow system uses more reagent than a discrete analyzer because a flow instrument is continuously pumping reagent through the system.
Discrete analyzers that measure the sample absorbance within the same container that the reaction occurred generate less waste than instruments that wash the vessel, or use a flow cell. In fact, adequately rinsing a flow cell requires significant rinsing between samples making the waste volume generated essentially equivalent to that of a micro-flow Segmented Flow Analyzer, or Low Flow Flow Injection Analyzer.
The discrete analyzer uses significantly less reagent, and generates significantly less waste than manual methods. This chart illustrates an unscaled down manual method using the exact volumes described in Standard Methods. The waste generated for the manual method does not take into account washing of glassware. As mentioned earlier, an analyzer that washes cuvettes or rinses a flow cell will generate more waste than indicated here.
Eliminate the possibility of contamination, or false positives
The discrete analyzer measuring the absorbance of a color reacted sample contained in individual cuvettes. Unlike flow analysis, there is no possibility of interaction between samples and unlike flow analysis; the user can visually observe the reaction product during and after analysis.
Using a discrete analyzer, the analyst can observe the reaction during color development and after the test is complete. The analyst 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 analyst the ability to visually examine and qualitatively guarantee the accuracy of his or her results.
A discrete analyzer dispenses, reacts, incubates, and measures all within the reaction cuvette without transferring to a flow cell. Analyzers that transfer to a flow cell are not “true” discrete analyzers, but instead, are hybrids between flow and discrete. The hybridization is done to achieve lower detection limits; however, the advantage of the individually contained reaction and absence of carryover is lost. In addition, since these analyzers require 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 discrete analyzers have reaction segments. Some analyzers do chemical reactions in a cuvette segment and then transfer the reacted sample to a flow cell. This type of analyzer is a hybrid of discrete and flow, and not a true discrete analyzer. A true discrete analyzer reacts and measures the sample within the optical cuvette. Some analyzers wash the optical cuvette between tests. Washing between tests enables more samples to be analyzed per cuvette; however, the washing cannot guarantee that there is no residual contamination that remaining after the washing process. Other discrete analyzers utilize disposable optical quality cuvettes.
Washing between tests enables more samples to be analyzed per cuvette; however, the washing cannot guarantee that there is no residual contamination not completely 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 optical quality cannot verify absence of chemical contamination.
Analyzers that use a flow cell still react samples in some sort of cuvette. It is the number of reaction vessels on the discrete analyzer that limit the number of tests that the discrete can run in a single walk away operation. If the discrete analyzer has 100 sample positions and 200 reaction cuvettes, then the analyzer can run 100 samples for 2 tests each. The discrete analyzer with the flow cell must rinse the flow cell between each sample, and rinse vigorously between 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 discrete analyzer with a flow cell. Also, consider that the flow analyzer generates no more waste than the discrete 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 between samples. Using disposable individual reaction cuvettes completely eliminates the possibility of contamination. For instance, the cadmium reduction nitrate test contains significant amounts of ammonia in the buffer reagent and phosphate in the color reagent. Using individual disposable cuvettes ensures that there is no contamination. Washing cuvettes, or using a flow cell, means you can never be sure.
Using disposable optical cuvettes is the only way you can guarantee no carryover between tests or samples. The concept is similar to use of disposable petri dishes, disposable pipette tips, and disposable hypodermic needles. The discrete analyzer easily and rapidly analyzes multiple tests on single sample solutions. Only disposable individually contained reactions ensure that there is no interaction between samples or tests.
Let the robot do your pipetting.
When you manually pipette samples you, hopefully, use a different pipette per sample. If not, you will at least rinse it in between samples, and possibly with sample prior to transferring your sample aliquot to the sample container. This is to avoid carryover between samples. A flow analyzer uses an auto sampler. The sampling probe immerses in the wash station rinsing the outside of the probe, and pulls wash solution from the station and into the analytical cartridge.
A discrete analyzer also uses a probe; however, it operates differently than flow analyzers. A discrete analyzer’s level detect mechanism ensures that the probe immerses into the sample or reagents no further than necessary to withdraw the required sample aliquot. The probe then washes itself on the outside at the wash station and pushes the sample or reagent out into the sample cuvette. Between dispenses, the probe pushes excess wash water out ensuring no carryover. In other words, unlike a flow system that only pulls sample in one direction, the sampling probe on a discrete analyzer is bidirectional pulling reagent and sample into its internal tubing only far enough to withdraw the correct volume and then dispensing it by pushing it out the other way.
The machine can think.
When doing a manual 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 discrete analyzer with level detection prevents this. The level detect mechanism is a capacitance detector that senses the difference between liquid and air. The discrete 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 usually adjustable by the user and is usually towards the bottom of the sample vial. On a discrete analyzer, the depth the probe immerses in a sample solution is a result of programming or instrument design. The depth sampled on the OI Discrete analyzer is determined 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 determined by the volume of the cup and the liquid level detected and withdraw a software-defined amount above 200 micro liters. In other words, the discrete analyzer samples from the top 300 micro liters of sample solution. The probe only immerses as far as it has to. This minimizes potential carryover contamination, and speeds the process. In this way dispensing and rinsing is fast and there is no sample or reagent carried to another on the sides of the probe.
When sampling from the top of the sample cup there is a risk of loss of a volatile analyte from the top of the solution 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 solution can be slowly lost from the top layer of sample solution into the lab air. This is especially evident with lower concentrations such as 10 ppb.
Gain of the analyte is possible as well. Ammonia is a common laboratory contaminant. Ammonia readily adsorbs into acidified solutions. It is possible for ammonia to be “pulled” from laboratory air into the sample solution. A flow analyzer would not as readily detect this loss or gain because it samples from the bottom of the sample cup.
There are some drawbacks
A discrete 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 develop color. In manual testing the reagents are added in open containers, however, the container shape can vary and the container can be capped during mixing, heating, and color reaction. When flow analyzers were first introduced one of the key advantages that gained its acceptance over manual methods was that reactions occurred enclosed within the tubing limiting its exposure to laboratory air. In this aspect, discrete analyzers are kind of a step backwards.
There are significant advantages.
Similar to holding a color developing reaction in its own container till it reaches a color maximum, discrete 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 useful in enzyme or reduction reactions where reaction rates are slow. A flow analyzer would require long delay coils resulting in very complex SFA chemistry manifolds. Often elevated temperature is used to speed reactions, but in some chemistry, there are limits to the maximum temperatures possible. Since discrete analyzer reactions are occurring in individually contained cuvettes, the time delay between reagent additions on discrete analyzers is limited only by software. This is a significant advantage over flow chemistry.
In manual 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 discrete analyzer. The dilutions can be preset during sample table entry if you know that the samples need to be diluted. Methods can be programmed such that they dilute every sample and standard all the time, or the instrument can be programmed so that over calibration, samples are diluted and re analyzed.
An analyst changes a manual or flow method from one to the next by memory, or by referring to the SOP. How well this particular analyst performs the procedure is dependent upon his mood, the time of day, his experience with the method, the availability of equipment, and many other unquantifiable variables. It is possible to obtain good results and bad results by the same manually performed method. A flow analyzer analyzes everything 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 multiple users results will vary. Extensive training and documentation is necessary to guarantee that results conform to good automated lab practices.
The discrete analyzer method is selected by mouse click when scheduling analyses on the sample tray. The method conditions do not change. In fact, assuming you have accurately calibrated your method the calibration is stored within the method. This means that an untrained analyst that only knows what buttons to press is able to obtain identical results to even the most experienced analyst.
Most analytes performed in an environmental compliance laboratory cannot be bench spiked. If the analyte requires a preliminary distillation, digestion, or extraction the spiking is done prior to the preliminary sample process. I realize 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 better seriously consider changing your SOP. Other parameters that can’t be spiked are those that are too high to spike within the matrix without preliminary dilution, such as Ca, Mg, Cl, SO4, and analytes like alkalinity that just are not spiked.
This shortens the list of potential analytes for the automatic spiking function to nitrite, phosphate, Sulfide, Chromium VI, and some others. On these, I defer back to the previous slide and ask if the potential error is worth the risk for so few tests.
Benefits of discrete analyzers include decreased reagent consumption, decreased waste generated, and ease of use among other things. The most significant advantage of the discrete analyzer, however, is that it can eliminate the traditional concept of routine analysis and allow you to run samples as you receive them instead of storing them until there is enough sitting around to make a flow or IC analysis worthwhile. If you take advantage of the calibration stability of the discrete analyzer, and accurately prepare a calibration that can then be used by almost any analyst in subsequent uses an added benefit is that the results are the same regardless of who uses the machine.
Think of those short holding time samples. The phosphate, the nitrites, the chromium VI, and residual chlorine. These analytes cause the environmental lab to stop everything 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 guarantee these tests followed the SOP? Instead of thinking of the discrete 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 discrete analyzer. Where you will save money and great effort is removing unnecessary strain from the flow analyzer and your analysts by performing the non – routine or “rush” tests on a discrete analyzer. It is possible for the sample login person to analyze samples as received for almost every colorimetric test that does not require 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 everything is to run on the discrete analyzer, then collect your samples in a vial that fits on the discrete analyzer. You no longer need to transfer liquid from container 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 discrete analyzer is that its built in features allow any analyst to get the same results every time. Discrete 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 discrete analyzers can be used effectively in your operation. Currently, the full power of discrete analyzers is limited by tradition and by regulation. Once we start to develop methods for discrete analyzers instead of using discrete analyzers to run methods developed for flow we will be able to see greater throughput, less variability, and lower MDL.