Every month we receive many questions and comments regarding phosphorus testing. Many people have trouble with this test. The procedures in Standard Methods are confusing, to say the least.
We estimate that about 50% of the problems are caused by confusion with the math, because of the volume changes within the Standard Methods' procedure.
After many years of wrestling with this problem, we have come up with a procedure which is adapted from Standard Methods, but removes the calculation problems by changing volumes so that the final volume is the same as the initial volume. (We estimate that 30% of the problems are due to running samples with concentration that exceed the linear portion of the "curve", and about 20% are due to poor lab technique, contaminated glassware, etc.)
Most people run this test in accordance with Standard Methods, using the Persulfate Digestion, followed by the Ascorbic Acid Procedure. This method has potential pitfalls which we will address here.
For those of you running the test for the first time, or with very limited experience, the most common problem is contaminated glassware. Wash the glassware well, using a non-phosphate containing detergent. Don't assume anything. Don't take anybody else's word, especially a salesman. READ THE LABEL! Even new glassware needs to be washed, because it goes through a washer before it is packed at the factory, and they use phosphate detergents!
Follow this by rinsing with 10% Hydrochloric Acid, then triple-rinse with deionized or distilled water (phosphate-free). DO NOT re-use the 10% Hydrochloric Acid.
Make your Combined Reagent fresh daily, as directed by Standard Methods. It should be a light straw or light yellow color. If it is blue, or shows any tingle of green (blue + yellow = green), throw it away and start over. You are wasting your time if you don't.
The next common problem is not having the proper pH just prior to adding the Combined Reagent. Make sure you add enough Sodium Hydroxide to just turn the solution light pink. Be sure to swirl after ever drop is added near the end-point. Then add one drop of 5N Sulfuric Acid. Swirl and make sure the solutionis water-white. If not, add another drop and swirl again. You may have to add several drops if you added too much Sodium Hydroxide previously.
After adding the combined reagent, line the flasks up in front of you, starting with the blank and continuing with increasing concentrations in sequence.
If this is your first attempt, don't be surprised if they look like this. You need to re-wash your glassware and start over. Don't bother turning on the spectrophotometer. If they look like this to you, the instrument will see them the same way.
Eventually they will look like this. Now you're ready to turn on the instrument to take readings.
- To warm up your spectrophotometer long enough.
- The blank contains distilled or deionized water PLUS all of the reagents added to the other standards and run through the entire procedure, like the standards. THE BLANK IS NOT JUST DISTILLED WATER!!!
People in Wisconsin and several other states are NOT permitted to use pre-programmed curves. Be sure to check with your state's regulatory agency. If this is the case, you need to measure the standards in the Absorbance mode, which is a better idea anyway, as the following will illustrate.
As you can see from the above graph, the results are only linear up to about 0.7 on the Absorption scale. This point will vary with different procedures and different instruments, but it will ALWAYS occur. You need to run the standards up high enough, at least once, to determine what the break-off point is for your procedure and your instrument. Once determined, you cannot use any "A" readings above this value. If you do so, your answers will be low. If you run several dilution's of a sample (which you always should, especially with blind and reference standards), you must discard all "A" readings above this value (DO NOT average them in)!!! If you did not have any "A" readings on the sample that were below this value, the sample needs to be diluted further and re-run from the start. There is no way to cut corners on this. I estimate that 50% of those who fail their reference standards, do so because of this phenomenon.
Last, but not least: Calculation errors. There is neither the time nor the space here to critique the Standard Methods procedure. Suffice it to say that if you think you understand the calculations, you probably don't. The best advice on this matter is to remember that the instrument only "sees" the solution(s) you put into it. The instrument does not know how much any of them were diluted. So you need to take all dilution's into account after you have read the "mg/l as P" from your graph. Double-check and triple-check your math!