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Paul Mack Sierra Club / RPG River Monitoring Program Feb 10, 2003
Standard Addition
Standard Addition is a technique that helps qualify dubious test results. However, before we discuss it in detail, let us define some new terms and review the concepts of STANDARD and BLANK.
New Terms Analyte - The chemical you are testing for. For example, when testing for Ammonia, Ammonia is the “analyte”. When a doctor takes a blood sample for a cholesterol test, cholesterol is the “analyte”. Interferences - Chemicals in the water that inadvertently interfere with our test. Method - The chemicals, procedures, and Hach machine settings which define a particular test. For example, there are many methods for testing Ammonia; the one we use is the “Nessler Method”. Any given method is accurate over a finite range of analyte concentration, from zero to some maximum value called a “ceiling”. Reagent - The Hach chemicals we add to the samples that make them change color. For example, when testing for Nitrate, the NitrVer5 powder pillow is the reagent. Standard Addition - A test technique for investigating suspicious test results. Titration - A method of determining the unknown concentration of an analyte in a solution, by adding to it a reagent of known concentration in carefully measured amounts until a reaction of known proportion is completed, as shown by a color change, and then calculating that unknown concentration. Titration provides the foundation for our test procedures.
What is a “standard”? In general, a standard is some official yardstick by which other things are measured. The dictionary definition is “something set up and established by authority as a rule for the measure of weight, etc.” For example, a scale company, by law, must use “standard” weights to calibrate its scales. These are expensive, extremely precise bronze weights that would be engraved with an official seal such as “1.00 U.S. pounds NIST-certified”.
We use similar standards in our water testing. The “standard solutions” in the large plastic bottles are guaranteed by Hach to contain exactly 1.00mg/L of an analyte (e.g. Ammonia).
What is a “blank”? In contrast, a blank represents nothingness. Its dictionary definition is “devoid of content”; in our use, it means “devoid of analyte”. It is nothing but pure water and reagent.
Thus, the BLANK calibrates the Hach spectrophotometer by establishing its zero-point, and the STANDARD verifies that the system (machine, chemicals, and test procedures) are all working properly. The BLANK and method ceiling establish the left and right boundaries of the system - the BLANK is the minimum (0.0) and the method ceiling is the maximum. Ideally, all samples should fall between these values (i.e. 0...ceiling); the STANDARD always will. Mathematically, this is expressed as BLANK ≤ sample ≤ method ceiling. Method ceilings are explained in more detail in the following pages.
Example In the following table, we compare the calibration of the Hach machine to that of a weighing scale, in a step-by-step manner. A scale was chosen for this example because it is analogous to the Hach tester.
Like the Hach tester, every scale (such as a bathroom, kitchen, or deli scale) has a zero point, a maximum point (“ceiling”), and a linear region in between; this is true for older mechanical scales as well as newer digital ones. Within that linear region, a scale will read the correct weight; outside that region, all bets are off. Ditto for the Hach.
In this example, imagine you are working the deli counter at Jewel. A customer brings a Tupperware container from home and asks you to fill it with six ounces of potato salad. Since the Tupperware itself weighs several ounces, you must think of some way to cancel out that weight, so that the customer will be charged only for the potato salad, not for the potato salad and Tupperware.
Procedure for using Hach tester Procedure for using a deli scale
- Place the BLANK in the tester and press ZERO button
- Place the blank (empty) Tupperware container on the scale and press ZERO button
- The tester should read “0.0” 2. The scale should read “0.0”
- Place the STANDARD in the tester 3. The customer doesn’t trust your scale, so she goes to aisle five and returns with a standard one-pound box of C&H sugar, which she asks you to pour into the Tupperware container
- The tester should read “1.0” 4. The scale should read “1.0”
- The tester is now ready to test river samples
- The scale is now ready to weigh potato salad
there are practical limits: if Sample Z contains 10,000 times as much Phosphorus as Sample Y, how can it be made 10,000 times bluer than Y?? It can’t, and so, Hach establishes a method ceiling for every test method. The ceiling is a limit imposed by the method components (machine, reagent, and procedures), and is different for every test method. The ceiling specifies the maximum amount of analyte that the system can accurately measure. Beyond that, things go nonlinear and measurements are no longer accurate. The following table lists the ranges for our four test methods:
Analyte Method Ceiling (mg/L) Test Range (mg/L)
Phosphorus 2.5 0 ... 2. Nitrate 4.5 0 ... 4. Ammonia 2.5 0 ... 2. Chloride 20.0 0 ... 20.
Thus, our reagents are not magic. They are simply chemicals which are known to change color very precisely and in proportion to the presence of some other chemical (the analyte). For example, Mercuric Thiocyanate reacts with Chloride to produce an orange color, while Nessler reagent reacts with Ammonia to produce a yellow color. Hach carefully chose these reagents and then designed the tester to work hand-in-hand with them.
Hach Spectrophotometer Our Hach tester is a “spectrophotometer”, a computer that measures the intensity of light in a given spectrum; that is, it measures the amount of color in a water sample. When you insert your sample bottle into the machine, a laser shines through the sample to a detector on the opposite side. The detector measures the amount of colored light that passed through the sample, and from that, the computer calculates the amount of analyte in your sample and displays it on the screen. For example, when you enter program #70 into the machine, you are telling it to test for Chloride, by measuring how orange your samples are. Actually, it does so using orange’s complimentary color - blue, which is why you must tune it to “455” (the wavelength of blue = 455nm 1 ). Likewise, when you enter program #380, you are telling it to test for Ammonia, by measuring the amount of yellow in your samples. Again, you tune the machine to “425”, because violet is the complimentary color of yellow, and the wavelength of violet is 425nm.
Interferences
Hach guarantees several key items: it guarantees that a Standard Solution contains exactly 1mg/L of a given chemical, it guarantees that a reagent will produce the correct color in the presence of a given chemical, and it guarantees that the machine will work properly with those items. However, Hach cannot guarantee that a reagent will respond to one and only one chemical. There are millions of different chemicals in the world (several hundred of which may be in the river), and it is impossible to design a reagent that will react to only one of them. So, while Hach has carefully chosen reagents which are unlikely to be disturbed by chemicals commonly encountered in surface water testing, it will occasionally happen, and this leads to the problem of interference.
1 1 “nm” = nanometer, which is one-billionth of a meter (0.000000001 meters)
An interference is a chemical in the water which inadvertently reacts with a reagent. How it exactly reacts with the reagent is unknown to us (we don’t really care, anyway); the important thing is that it will interfere in some way with the test by adding or subtracting color, and thus, it will ruin the results.
For example, we know that Mercuric Thiocyanate reacts with Chloride in a well-defined manner. What we don’t know, however, is what else Mercuric Thiocyanate reacts with. For all we (or Hach) know, it might also react with zinc (or calcium or even something more complex like motor oil) and thus, if your river sample contains both zinc and Chloride, the sample might turn too orange, and the Hach meter will unwittingly display a false reading. Note that this will occur regardless of how well you prepared the BLANK and STANDARD!
That said, note that interferences are rare, and it would be even rarer for more than one interference to be in the river. Thus, if data from more than one test appears bad, then interferences are not to blame. For example, if your Phosphorous and Chloride results are questionable, then the problem is not with interferences.
Conversely, if the river contains an interference for one of the reagents, it will not likely interfere with the other reagents. For example, if it is found that the river contains a chemical that is interfering with the Nitrate test, Ammonia, Phosphorous, and Chloride tests will not be affected and their data will be fine.
Hach has published a list of chemicals that are known to interfere with its products. An abridged list is provided in the appendix at the end of this report.
So, how do you uncover the problem of a hidden interference? Through the use of a Standard Addition.
Standard Addition
The technique of Standard Addition consists of adding a known amount of a Standard Solution to a river sample, and then testing it as usual.
If the result does not match that which you calculated, then there may be an interference in the river that is ruining your test. There is nothing you can do to correct this, so note it on the worksheet and logbook, and then contact the test coordinator.
On the other hand, a result that agrees with your calculation says nothing about the source of bad data; it simply rules out interferences. By process of elimination, though, it does suggest that the deviation might be due to natural variation or careless mistakes.
Therefore, the Standard Addition technique only indicates if an interference might be to blame for bad test data. (To a lesser extent, it can also help identify spoiled reagents, a faulty machine, or impure distilled water.) It cannot, however, determine if bad test data was caused by natural variation or careless mistakes.
liter and a milligram is one-thousandths of a gram (to get a notion of grams and milligrams, check the label on a bottle of aspirin or vitamins).
Dimensional Analysis, then, is the mathematics of dimensions; i.e. converting from one unit to another. This is a key component of the Standard Addition calculations. If you can get past that hurdle, you will be well on your way to understanding and using Standard Additions.
Performing Standard Addition Standard Addition consists of three distinct steps: calculate the theoretical concentration, measure the actual concentration, and then compare the two results.
- Calculate Theoretical Concentration of Spiked Sample Using dimensional analysis, reduce concentration (mg/L) to weight (mg), to simplify the Standard Addition calculations (this is the least messy and most intuitive manner of understanding the math involved). Then, calculate the total weight of the analyte (i.e. river water + Standard Addition) in the small bottle. Finally, scale that value back up to mg/L. Note that the concentration value used here is from the bad data you are attempting to validate.
- Measure Actual Concentration of Spiked Sample To a clean, dry bottle, add the normal amount of river water (note that this is a “fresh” sample from the mason jar). Then “spike” it with the proper amount of Standard Solution (taken from the large plastic Hach bottle, not from your little STANDARD!) Then, test it as usual - add the normal reagents, wait for the specified time, and place it in the Hach machine for measuring. This step is analogous to the Mrs. Dash example.
- Compare the Results from #1 and # If the measured result differs from that calculated by more than 10% or so, it indicates that your original data was corrupted by interferences.
For whatever reason, if you choose not to redo the test, then your original “bad” data is recorded on the worksheet and logbook. The Standard Addition data from #1 and #2 is for diagnostic purposes only - it is not to be recorded!!!
In summary, the technique of Standard Addition consists of three distinct steps:
- do the math
- spike a fresh river sample and then test it as usual
- compare the results of #1 and #2, and then discard them
Thus, this technique does not attempt to deliver the correct test values; rather, it is simply a Yes/No indicator as to whether your original test was ruined because of interferences in the river water. For this reason, we “discard” the results.
The amounts of river water and Standard Solution must be precisely measured, and are given in the following table:
Analyte Container
Add this much...
Multiply
2
Hach
reading by
River
Water
Std
Soln
Distilled
H2O
1
Phosphorus 10ml glass bottle 2.0ml 3.0ml 5.0 ml 2 Nitrate 25ml plastic bottle 5.0ml 5.0ml 15.0ml 2. Ammonia 25ml plastic bottle 15.0ml 10.0ml none 1 Chloride 25ml plastic bottle 1.0ml 5.0ml 3 19.0ml 4.
notes: (^1) no need to actually measure distilled H 2 O, just add river water & std soln and then fill to the line as usual
(^2) scaling factor compensates for dilution:
river water + std soln + distilled water river water + std soln (^3) this is from the diluted standard solution you mixed in the 500ml flask
Standard Addition - Phosphorus
- Calculate Theoretical Concentration of Spiked Sample Given: From the table on page 8, we see that our 10ml glass bottle will contain 2.0ml of river water, 3.0ml of Standard Solution, and the remainder (5.0ml) is distilled H 2 O.
A. Calculate the actual amount of Phosphorus in the river sample that yielded your bad test data. As an example, let’s use the “bad” reading (pink row) from my worksheet:
Sample Amt of Sample Reading Result WB2.0 2.0ml 1.41 × 5 = 7.05mg/L
Express this concentration as mg/ml:
7.05 mg L ≡^
7.05 mg 1000 ml =^
0.00705 mg ml
Calculate actual amount of Phosphorus:
0.00705 mg ml × (^2) ml = (^) 0.0141 mg
B. Calculate the actual amount of Phosphorus in 3.0ml of Standard Solution. Hach guarantees its concentration is 1mg/L.
Express this concentration as mg/ml:
1.0 mg L ≡^
1.0 mg 1000 ml =^
0.001 mg ml
Calculate actual amount of Phosphorus:
0.001 mg ml × (^3) ml = 0.003 mg
C. Add these two results to get total amount of Phosphorus in the glass bottle.
0.0141 mg + .003 mg = 0.0171 mg
D. Convert this back to concentration (mg/L), noting that only 5ml of liquid contributes to the total in step C (the 5ml of distilled H 2 O contributes nothing).
0.0171 mg 5 ml ×
=
0.0171 mg 1000mlL^ 0.005 L^ =^ 3.42 mg / L
Thus, the theoretical concentration of our spiked sample should be 3.42mg/L.
Standard Addition - Phosphorus (cont’d)
- Measure Actual Concentration of Spiked Sample Note that, with the exception of step B, this procedure is exactly the same as a normal test.
Procedure: A. Add 2.0ml of river water to a clean, dry 10ml glass bottle. Note that this is a “fresh” sample from the large mason jar. B. Spike it! Add 3.0ml of Phosphate Standard Solution (from the large plastic 500ml Hach bottle). Use a new pipette for this. C. Fill to the 10ml line with distilled H 2 O. D. Add the PhosVer3 powder pillow, swirl, and wait two minutes. E. Place the sample bottle into the Hach tester and note the reading.
- Compare the Results from #1 and # Do the results from step 1D. and 2E. match? If they differ by more than 10% or so, it suggests that your original data may have been corrupted by interferences.
In my own example, the Hach machine reported that my spiked sample was 1.81 (green row on worksheet); per the table on page 8, multiplying by two yields 3.62mg/L, which is close to the theoretical result in step 1D. Therefore, there were no Phosphorous interferences in the West Branch DuPage River, so something else is to blame for my bad data.
Finally, note that results you receive in steps 1D. and 2E. are for a spiked sample, not a real river sample. Therefore, regardless of what you concluded in step 3, the results do not reflect the amount of Phosphorus in the river, and thus, are not recorded in the log book.
Standard Addition - Nitrate (cont’d)
- Measure Actual Concentration of Spiked Sample Note that, with the exception of step B, this procedure is exactly the same as a normal test.
Procedure: A. Add 5.0ml of river water to a clean, dry 25ml plastic bottle. Note that this is a “fresh” sample from the large mason jar. B. Spike it! Add 5.0ml of Nitrate Standard Solution (from the large plastic 500ml Hach bottle). Use a new pipette to do this. C. Fill to the 25ml line with distilled H 2 O. D. Add the NitraVer5 powder pillow, shake for one minute, and then wait five minutes. E. Place the sample bottle into the Hach tester and note the reading.
- Compare the Results from #1 and # Do the results from step 1D. and 2E. match? If they differ by more than 10% or so, it suggests that your original data may have been corrupted by interferences.
In my own example, the Hach machine reported that my spiked sample was 2.0 (green row on worksheet); per the table on page 8, multiplying by 2.5 yields 5.0mg/L, which is identical to the theoretical result in step 1D. Therefore, there were no Nitrate interferences in the West Branch DuPage River, so something else is to blame for my bad data.
Finally, note that results you receive in steps 1D. and 2E. are for a spiked sample, not a real river sample. Therefore, regardless of what you concluded in step 3, the results do not reflect the amount of Nitrate in the river, and thus, are not recorded in the log book.
Standard Addition - Ammonia
- Calculate Theoretical Concentration of Spiked Sample Given: From the table on page 8, we see that our 25ml plastic bottle will contain 15.0ml of river water, 10.0ml of Standard Solution, and no distilled H 2 O.
A. Calculate the actual amount of Ammonia in the river sample that yielded your bad test data. As an example, let’s use the “bad” reading (pink row) from my worksheet:
Sample Amt of Sample Reading Result WB2.0 25.0ml 0.31 0.31mg/L
Express this concentration as mg/ml:
0.31 mg L ≡^
0.31 mg 1000 ml =^
0.00031 mg ml
Calculate actual amount of Ammonia:
0.00031 mg ml × (^15) ml = (^) 0.00465 mg
B. Calculate the actual amount of Ammonia in 10.0ml of Standard Solution. Hach guarantees its concentration is 1mg/L.
Express this concentration as mg/ml:
1.0 mg L ≡^
1.0 mg 1000 ml =^
0.001 mg ml
Calculate actual amount of Ammonia:
0.001 mg ml × (^10) ml = 0.010 mg
C. Add these two results to get total amount of Ammonia in the plastic bottle.
0.00465 mg + .010 mg = 0.01465 mg
D. Convert this back to concentration (mg/L), noting that all 25ml of liquid contributes to the total in step C.
0.01465 mg 25 ml ×
=
0.01465 mg 1000mlL^ 0.025 L^ =^ 0.586 mg / L
Thus, the theoretical concentration of our spiked sample should be 0.586mg/L.
Standard Addition - Chloride
- Calculate Theoretical Concentration of Spiked Sample Given: From the table on page 8, we see that our 25ml plastic bottle will contain 1.0ml of river water, 5.0ml of the diluted Standard Solution you mixed in the 500ml flask, and the remainder (19.0ml) is distilled H 2 O.
A. Calculate the actual amount of Chloride in the river sample that yielded your bad test data. As an example, let’s use the “bad” reading (pink row) from my worksheet:
Sample Amt of Sample Reading Result WB2.0 1.0ml 6.1 × 25 = 152.5mg/L
Express this concentration as mg/ml:
152.5 mg L ≡^
152.5 mg 1000 ml =^
0.1525 mg ml
Calculate actual amount of Chloride:
0.1525 mg ml × (^1) ml = 0.1525 mg
B. Calculate the actual amount of Chloride in 5.0ml of the diluted Standard Solution, whose concentration is 10mg/L.
Express this concentration as mg/ml:
10.0 mg L ≡^
10.0 mg 1000 ml =^
0.01 mg ml
Calculate actual amount of Chloride:
0.01 mg ml × (^5) ml = 0.05 mg
C. Add these two results to get total amount of Chloride in the plastic bottle.
0.1525 mg + .05 mg = 0.2025 mg
D. Convert this back to concentration (mg/L), noting that only 6ml of liquid contributes to the total in step C (the 19ml of distilled H 2 O contributes nothing).
0.2025 mg 6 ml ×
=
0.2025 mg 1000mlL^ 0.006 L^ =^ 33.75 mg / L
Thus, the theoretical concentration of our spiked sample should be 33.75mg/L.
Standard Addition - Chloride (cont’d)
- Measure Actual Concentration of Spiked Sample Note that, with the exception of step B, this procedure is exactly the same as a normal test.
Procedure: A. Add 1.0ml of river water to a clean, dry 25ml plastic bottle. Note that this is a “fresh” sample from the large mason jar. B. Spike it! Add 5.0ml of the diluted Standard Solution from the 500ml flask. C. Fill to the 25ml line with distilled H 2 O. D. Add the Mercuric Thiocyanate, etc. and then wait two minutes. E. Place the sample bottle into the Hach tester and note the reading.
- Compare the Results from #1 and # Do the results from step 1D. and 2E. match? If they differ by more than 10% or so, it suggests that your original data may have been corrupted by interferences.
In my own example, the Hach machine reported that my spiked sample was 7.9 (green row on worksheet); per the table on page 8, multiplying by 4.167 yields 32.92mg/L, which is close to the theoretical result in step 1D. Therefore, there were no Chloride interferences in the West Branch DuPage River, so something else is to blame for my bad data.
Finally, note that results you receive in steps 1D. and 2E. are for a spiked sample, not a real river sample. Therefore, regardless of what you concluded in step 3, the results do not reflect the amount of Chloride in the river, and thus, are not recorded in the log book.
