# Test Tube Cleaning



## rjordan393 (Nov 23, 2012)

I have a suspicion that my glass test tube for PO4 testing have been contaminated by the PO4 reagent and may not be salvageable. The test tube was rinse well and scrub with a small bottle brush after every test. But it may not be enough. Many years ago, a chemist recommended using muriactic acid to clean test tubes but I failed to follow up on that advice. Yesterday, I tested and I got a 2 ppm reading. So today, I added enough KH2PO4 that should have brought it up to 6 ppm. When I tested again, the test read 16 ppm. I am using a "Hach" test kit and do not doubt the quality. So I added some acid to the tube and shaked it a bit and let it sit for about 3 minutes. I noticed it took on a slight blue tint. Afterwhich, I dumped it and rinsed it well and used the small bottle brush on it. I tested again and got the same result (16ppm). My conclusion is that the reagent residue does not come off easily if the tube is not acid washed immediantly after testing.
I also checked my dose with the Fertilator and Wet's calculator and they were the same. I am also looking into the possibility that my KH2PO4 is coming out of solution. It contained 36 grams to 500 ml. This would increased the strength as the contents get lower in the bottle.
Tommorow, I will test again using a brand new test tube to see if this confirms my conclusion.
Your thoughts?


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## Tugg (Jul 28, 2013)

Are you mixing with other ferts, or is this just a single dosing bottle with KH2PO4? Make sure you're using distilled water. With a solubility of 22.6g/100mL, you shouldn't have any precipitation.

Our typical macro NPK mixes all have K as the cation (KNO3, KH2PO4, K2SO4). So a Macro mix is fine. If you add Ca, Mg, or other traces, you'll precipitate.

If Phosphate is mixed with pretty much any other cation other than ammonium (NH4) or the group 1A metals (Li, Na, K, Rb, Cs), it will form precipitates. So with tap water, it will react with the calcium and magnesium.


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## JeffyFunk (Apr 6, 2006)

The phosphate analysis procedure is based off of the reaction of phosphates with an antimony and molybdenum complex, which is reduced with ascorbic acid to produce the characteristic blue color. Because the color reagents are metals, it does make sense that the containers should be washed with a weak acid solution (like 1:9 HCl or HNO3) since metals are most soluble in acidic solutions. (Hot dilute HCl is even better for rinsing...) 

Looking up the analysis procedure in the Standard Methods book, positive interferences include arsenates (AsO4---) and silica. That said, a 100 ppm Si interference will cause the phosphate analysis to be biased high by only 0.025 ppm PO4-P. (That's in units of P - i don't have the conversion of P to PO4 off the top of my head). Maybe you need to use a plastic test tube for the Phosphate analysis? 

You might also want to soak your cleaning brush in an acid cleaning solution as well ... How do you know the brush isn't the source of the contamination? Also, check your cleaning solution (soap) - some soaps use phosphates in them, which could be another source of contamination.


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## rjordan393 (Nov 23, 2012)

I use distilled water for my individual fertilizers. Another PO4 test using a new test tube indicated I have 13 ppm PO4 in my 75 gallon tank. I had about 2 ounces left in the KH2PO4 container and I dumped it out and rinse the container with tap water. This time I added 43 grams of KH2PO4 to 500 ml of distilled water using labware to measure out 500 ml. I checked the calibration of my scale and it was off by 0.8 grams or 0.0186 per gram error. I do not think that is enough to cause the high error I am getting.
So to check the accuracy of Wet's calculator, I made up a new batch of KH2PO4 in my 500 ml container using 43 grams. According to Wet's calculator, 1.5 ml of it, dosed in 1 gallon of water should give me 23.78 ppm. But my high range test procedure using the HACH orthophosphate kit indicates 40 ppm after multiplying by 10 as indicated in the instructions. 
I wonder if its possible that my source water has a high level of PO4, The average on my county water report is 0.15 ppm.


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## JeffyFunk (Apr 6, 2006)

It's certainly possible to have high phosphates in your tap water - i know that i do. And if you really want to have the accuracy of your results checked, you could always send them to me and i can check them in the lab. Phosphate analysis is one of the easier tests to do.


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## rjordan393 (Nov 23, 2012)

I'll test the source water. My kit was testing PO4 normally for at least 2 tests on the low range scale at 1 to 2 ppm on both tests. Now all of a sudden when still using the remainder from the old KH2PO4, the PO4 went to 16 ppm and today down at 13 ppm in the tank. On my next water change, I think I will have a better idea of whats going on and I will does only 25% of Wet's recommended dose in ml for my tank to see where this leads me.


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## rjordan393 (Nov 23, 2012)

The source tapwater is 0.8 ppm PO4. So now I am looking at the chemcial itself. So using 1 gallon of tapwater and I dry dosed 0.05 grams (using a jewelers scale and calibrated) to it and according to the fertilator and Wet's calculator the result in my PO4 test should be 9.22 ppm. But the test read 44 ppm. This is simular to a result I got when I tested my 500 ml solution in 1 gallon of water using 1.5 ml. It appears something happened to the KH2PO4 which is stored in a ziplock plastic bag and placed amongst other fertilizers in a heavy duty vinyl bag.
So the question is: How can a fertilizer gain concentration? Or can the fertilator and Wet's calculator be wrong? I am perplexed.


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## Tugg (Jul 28, 2013)

I really don't like the idea of using tap for a baseline. I would get a gallon of distilled. It's only like $1.

If you repeated the results with distilled, then I would look at the test kit being no good. I highly doubt the simple salt can go bad. It's hygroscopic so perhaps it could hydrate some, but that would give a lower reading, not higher.

If it's caked up or moist, you could bake it in the oven on a low setting for a short while. But like I sad, I doubt that it's the salt. I'm guessing the Tap or the Test Kit.


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## rjordan393 (Nov 23, 2012)

Thanks Tugg.
You know, I should have mentioned that the test kit measures orthophosphate and I know that all aquarium test kits measure phosphate. How much of a differance does this make. I should kick myself if it does.
If the test kit is suspect, then the Hach company has a lot of explaining to do.


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## Tugg (Jul 28, 2013)

This is getting past my chem skills comfort zone, but here is some speculation that hopefully JeffyFunk can comment on.

When the salt dissolves, the two H+ could be dissociating and increasing the acidity. Perhaps the addition of the KH2PO4 is allowing additional orthophosphate to be released from the tap with the increased acidity.

You could test the theory by taking a phosphate test of your tap, then adding some vinegar _(acetic acid, CH3COOH has no P in it)_ to acidify it, and then take another reading of the phosphate.

Again... speculation and shooting in the dark on my part.


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## rjordan393 (Nov 23, 2012)

I wait to see if Jeff has a comment on this.


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## JeffyFunk (Apr 6, 2006)

From Standard Methods: Phosphates can exist in several possible forms: orthophosphates, condensed phosphates (pryo-, meta- and other polyphosphates) and organically bound phosphates. 

Orthophosphates are simply the unpolymerized or simplest phosphate unit, PO4---. That is the phosphate species most colorimetric tests analysis for, including the Hach Test. BTW, Which Hach test are you using? I'm guessing PhosVer 3 (Ascorbic Acid)?

...

According to my analytical textbook, the second pKa of the H3PO4 is ~ pH 7.144. This means that the salt KH2PO4 is actually slightly basic. 

...

Here's my personal take on all of this. First of all, i'm not a fan of Hach in general. Most of their test kits are nothing more than classical water chemistry packaged in such a way that you have to use their 'kits'. Since i actually work in a lab, it makes no sense for me to buy their 'kits'. That said, i also have hundreds or thousands of dollars in analytical equipment to support my analysis procedures and to verify that things are working correctly. Not everyone has that luxury. (Also, several of their tests are certified for use by the EPA. If they weren't accurate, do you really think they would be acceptable for environmental compliance?)

Here's a good example of what i mean by that. Just because you have a sensitive balance doesn't mean jack. You can calibrate it? Great, but it's still meaningless. How do you know that your balance is ACCURATE? Do you have your balance certified every year? Do you have verification weights to prove that you're able to measure things accurately? How do you know that your calibration weight is correct? Do you have an internal calibration weight? That's probably more accurate, but still no guarantee that's it's accurate. 

Unless you are using ACS or analytical quality reagents, certified volumetric pipettes and glassware, certified standards and secondary solutions, how can you have any confidence in your analysis results at all? Home test kits are great for getting a good estimate of your water chemistry since they are based off of classical water chemistry, but i think it's very naive to say it's all Hach's fault. I would credit you for finding issues with your procedure and questioning your results, but without the backing up of your results from an accredited laboratory (or at least a bunch of other test kits using certified reagents and standards), i think you're going to have a hard time going to the company and complaining.


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## rjordan393 (Nov 23, 2012)

I am using the Stannaver Method for PO4. The low range is 0 to 4.4 ppm and the high range is 0 to 44 ppm. The two scales I use are in use for 18 months and have calibration weights. I see what your saying but I am not after pinpoint accuracy. I purchase the Hach PO4 and Nitrogen test kits just to get a bit more accuracy then the hobby kits. I been making my own solutions of my fertilizers and I never seen a problem such as PO4 reading going as high as 40 to 44 ppm until recently. Prior to my post, I was running my tank at 1 to 2 ppm using the same solution containing 43 grams of KH2PO4 to 500 ml and I adjusted my tank concentration to 2 ppm after 7 days without no problem. Then while seeing some recommendations to get rid of greenspot algae by increasing the PO4 concentration; is when I decided to raise it to 6 ppm. I used Wet's fertilizer calculator to determine the amount to use. Then when it was time to test, The first day it was reading 16 ppm, the second 13 ppm and now it is probally around 10 ppm. Then when I tested my KH2PO4 by dry dosing 0.05 grams to 1 gallon of tap water which has a PO4 level tested at 0.8 ppm, my reading is 40 to 44 ppm.
The reading should have been 9.22 ppm.
If I do not solve this, I will rely on dry dosing KH2PO4 but at much less then the recommended dose and then test to see where I am at and go from there. Maybe the scales are at fault.


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## JeffyFunk (Apr 6, 2006)

What is the stannaver method? I can't seem to find that method in the Hach catalog or Water analysis handbook. According to their Water Analysis Handbook (Which is free, btw - and details all of their methods), none of the P analysis methods utilize a Sn reagent or anything of the sorts. 

Which of the following are you using? They list 3 methods: 

Amino Acid Method: In a highly acid solution, ammonium mobybdate reacts with orthophosphate to form molybdophosphoric acid. This complex is then reduced by the amino acid reagent to yield an intensely colored molybdenum BLUE compound. Test results are measured @ 530 nm. 

Molybdovanadate Method: Orthophosphate reacts with molybdate in an acid medium to produce a mixed phosphate/molybdate complex. In the presence of Vanadium, a YELLOW molybdovanadophosphoric acid complex is formed. Test results are measured @ 430 nm. 

PhosVer 3 (Ascorbic Acid) Method: Orthophosphate reacts with molybdate (and Sb) in an acid medium to produce a mixed phosphate/molybdate (and Sb) complex. Ascorbic acid then reduces the complex to produce an intense BLUE compound. Test results are measured @ 880 nm. 

My experience has been with method 3, the Ascorbic Acid Method, since this is the method listed in Standard Methods. 

I agree w/ your overall assessment of the problem that things were fine and analyzing okay until recently, with no change in your dosing regime. I would suspect the analysis results as well. Contamination is always on my mind @ work and i think you're on the correct path. 

I would doubt the scales would suddenly go bad, though. Unless it's some sort of electronic failure, though, in which case you can usually tell right away that something is amiss. And i would think that a bad scale would result in a systematic error, where all the results were off, not just one test and not just suddenly.


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## rjordan393 (Nov 23, 2012)

MY mistake. It is called Stannous method. The Hach catolog number is 147500 for the kit. If you imput that number in their search box, the product comes up and then click on parameter/reagent. You will see that it says Stannous method. Here are the chemicals that came with the kit.
Phosphate 2 reagent, catolog # 1061-99 contains Potassium sulfate & Stannous Tartrate.
Ammonium Molybdate reagent, Catolog 110-32H
Deionized Water, Catolog 272-42

Yesterday, I think I may have found two problems with my scales.
The jewelers scale can read to 3 decimals points (0.000). When I start to add a tiny amount to it, it does not indicate anything on the scale until I add a bit more. This explains why 0.05 grams to 1 gallon of water test out as 40 ppm. The real scale reading was higher then what was indicated. I'll have to check it further to see if there's some residue effecting the reading. The capacity is 20 grams, the resolution is 0.001 gram and the tolerance is + or - 0.003 grams. 

The larger scale, I believe may have a simular problem. Its capacity is 300 grams X 0.1 gram.
So one of the things I will do first is to vacumn both scales and then change the batteries even though there is no indication of low battery power.


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## Tugg (Jul 28, 2013)

I don't suppose you have anything that can accurately hold 20Gs? Extra tank perhaps. Then you could fill it and add 1g. The larger the standards, the more accurate it should be. It would be easier than fighting with hundredths of a gram.


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## rjordan393 (Nov 23, 2012)

Your right Tugg.
I was trying to do too much with my small jewelers scale to determine accuracy. I intend to do something simular to what you suggest. On the next water change, I will test right after. Then determine my parameter adjustments. Then about 30 minutes after dosing, I will test again to see how much my dose has effected the tank. It will take time, but if my scales are off that bad, then I'll go back to using teaspoons and dry dose.


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## JeffyFunk (Apr 6, 2006)

Okay, it took a little bit of looking, but i found it. The Stannous method is not listed in the Hach Water analysis handbook but a similar test is listed in Standard methods, SM 4500-P D (Stannous Chloride Method). Several things that stood out in my mind (1) Chloride interferes @ 75 ppm and (2) Rate of color development and intensity of color depend on the temperature of the final solution where each 1oC increase producing about 1% increase in color. Hence, hold samples, standards, and reagents within 2oC of one another and in the temperature range of 20-25oC. Also, the color fades over time so be sure to not wait too long to measure the concentrations. 

Tugg, the idea of using an aquarium to make up a standard solution with the premise that 'larger volumes have less error' is crap. No offense directed at you, but i see this stupid argument made all over the internet (esp by Tom Barr) and i just don't understand it. 

Here's how i believe this idea came about. If you are dealing with VOLUMETRIC glassware, then as the volume of the flask increases, the absolute error increases, but the relative % error decreases. Basically, this absolute error can be attributed to the error associated with reading the MENISCUS, which increases with volume as the diameter of the volumetric flask increases. So when you are doing your calculations for concentration, which is a multiplication function, the error analysis calculation is to use relative % error. So what has the lowest relative % error? The largest VOLUMETRIC flask. 

Now, what is the error associated with an aquarium? Answer? YOU HAVE NO IDEA! There is no meniscus to fill to, you have no idea what the actual volume is! How do you know you're reached exactly X gallons? And if you're dumb enough to think for a second that the actual volume is what the manufacturer states (i.e. 20 gallons = 20.00000... gallons)... well, i think we get the idea (and if you don't, then you probably wouldn't understand anything i've written in this post anyway...). 

Now that's the problem with the volume issue (which is believe is the largest source of error), but let's look at the mass issue. Generally speaking, you are correct in that if X g > Y g, then X should have a lower relative % error. But, you have to take into consideration what the error is of X & Y, which is related to your scale's sensitivity. 

rjordan393, i believe you are correct to look at your scale and consider the possibility that it is 'sticky', which would present itself as a problem more at the low end your analytical range. I have to 'tap' my balance several times in order to be sure that it's stabilized on a value and won't jump around anymore. That said, the only way to verify the low end of your analytical range would be to weigh a low level standard and see how accurate your scale is. (In our laboratory, we have to verify our analytical balance (with a sensitivity of 0.0001 g) with a 20 mg weight (0.0020 g) and a 100 g weight (100.0000 g).


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## Tugg (Jul 28, 2013)

Don't worry about me. I'm pretty tuff to offend.  I *love* a good discussion, and people telling me I'm wrong is the best way to make me learn. Also, by no means would I EVER trust the listed volume of an aquarium manufacturer. However, I would trust my ability to measure the internal dimensions of a rectangular box and calculate the volume with a moderate degree of accuracy.

You also have to remember, most home hobbyists' tools are crude to begin with. Most of us don't have graduated cylinders, we're using a milk jug. There is no scale to measure a meniscus against, we're just filling it back to about an inch from the top and calling that a gallon. Not very accurate. For the aquarium, assuming I could only accurately measure to within just half a cm, I'm still not likely to be more than 1/2 to 1 gallon off. That's about 5% on a 20 gallon. If I'm using a 1 gallon milk jug to begin with, I'm probably dealing with the same relative error measuring that single gallon if not more. So I would call the error in the solvent to be a wash. In either case, crude hobbyist tools probably leave us at around 5%. If I'm using the milk jug to fill the tank, then I may also get a small sense increased accuracy as it should take 20 jugs to fill to my 20 gallon mark.

However, with the larger standard, the error of my scale measuring the solute becomes less of an issue. If the scale measures .001g +/- .003g and I need .05g, I'm dealing with a much higher relative error (6%) than if I measured 1.00g on the same scale (0.3%). In this case rjordan393 has a nicer scale. On a common scale that only does 0.1g resolution you can't even make the smaller standard. You would need to make a dosing solution, and then add a sample of that to the gallon.

When I made my 4dKh solution for my drop checker I used the method of dosing high, and then repetitively diluting the solution by dumping half and adding distilled. Crrrrrrrrrude  But we work with what we have.


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## JeffyFunk (Apr 6, 2006)

Tugg said:


> However, with the larger standard, the error of my scale measuring the solute becomes less of an issue. If the scale measures .001g +/- .003g and I need .05g, I'm dealing with a much higher relative error (6%) than if I measured 1.00g on the same scale (0.3%).


This i agree with. However, a solution is only as precise as it's least accurate factor - in this case, the volume measurement. My point is that people don't take the volume measurement into consideration and that's equally as important when making a solution. In this case, it's even more important because the volume measurement becomes the limiting factor because it's the least accurate value in the concentration calculation. Comparing volumetric glassware to an aquarium is silly.


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## rjordan393 (Nov 23, 2012)

Thanks Jeff & Tugg,
Today is water change day. I will soon know if I will continue to use my scales. I intend to test after the change and adjust my parameters by dry dosing using + or - teaspoons and then test again. My results should be close to the calculations. Then when its time to dose again, I will have a new batch made up in PO4 using 43 grams of KH2PO4 in 500 ml of distilled water. I have volumetric lab glassware to measure the water. But this time I will dose only 25% of what Wet's calculator says, in case there is a large error Then I will know from the result whether my large scale is good enough for hobby purposes.


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