Measuring LR NO3 with Hanna ULR Phosphorous photometer

[amebcia]

Since many of us already have the Hanna ULR Phosphorus photometer why not to use it for measuring NO3 as well

The Hanna ULR Phosphorus photometer utilizes 525nm (green) light so can be used in conjunction with any other chemical test that tints a water sample blue or red (absorbs green). The only missing part is the calibration.

Below you can find instructions for measuring NO3 using the API NO3 test, but it can be easily adopted for most of the popular NO3 tests like Salifert, Nutrafin, JBL, Elos etc. It should also work fine with Hanna HI3874 ($30/100 tests) and HI93728-01 ($50/100 tests) reagents.

Instructions for API NO3 test below. I estimate it to be 10% +/- 1ppm accurate.
1. Fill in the original Hanna bottle with 1ml if aquarium water and 9ml of quality RO or distilled water (the photometer is very sensitive).
2. Add 20 drops of API reagent 1
3. Shake the sample for 2min keeping the bottle in a closed hand to warm it up.
4. Put the bottle in the Hanna ULR Phosphorus photometer and take it through step 1 (zeroing).
5. Remove the bottle and add 20 drops of API reagent 2.
6. Shake the sample for 1min keeping the bottle in a closed hand to warm it up (to speed up the chemical reaction)
7. Long press the button on the Hanna photometer to take it to step 2. 3min counter should start.
8. Do not put the bottle back in the photometer yet. Keep shaking it in a closed hand.
9. When the Hanna photometer timer gets down to 30s put the sample in and close the lid.
10. Wait until the timer gets to 0 and a measurement is made.
11. Divide the reading by 10. For example 10 "“ 1ppm NO3, 100 "“ 10ppm NO3, 199-19.9ppm NO3, 200- 20+ppm of NO3.

It turned out that API NO3 test gives a linear response, so to get the NO3 concentration it is enough to divide the reading by 10. Other reagents may have non-linear response so conversion may be a bit less straight forward. Also the required aquarium to RO water mix ratio may have to be different.

 

[watchguy123]

I like this. It certainly seems reasonable that a colorimeter should be adaptable to different test kits with colored results once the dilution factor is figured out. Assuming of course, as stated that the results are linear. And since I hate reading color charts, this may very well turn out to be phenomenal. But how did you determine the dilution and correction calculation. Was it a trial and error or what. And how are you sure of the linear change in color.

I am looking forward to trying this and comparing it to results using the api as instructed by the manual. I also would love the Red Sea phosphate dilution factors--imagine how cool it will be to no longer have to use that lousy powder in foil because I'm never sure of whether all the powder made it into the test tube.

Would like to see if others have found the results reliable and reproducible with this method. This is so potentially powerful to be able to use an inexpensive test kit (API) and hopefully get the advantage of a colorimeter from Hanna to lead to precise and reproducible results. Can't wait to try it and report back

 

[amebcia]

I got your PRV message, but guess what. When I clicked on "Send" the following message showed up "Sorry, you can only send messages to the RC Moderator group until you have 10 posts." :fun2:

To answer your questions one by one.

How many drops
API uses 10drops on a 5ml sample. Hanna bottle is 10ml, so 20drops instead of 10.

Dilution ratio.
A bit of background first. My goal was to test in the range of 0-20ppm NO3 so to start with I mixed a 20ppm NO3 calibration solution. Used 99% pure KNO3, deionized water and a digital scale with accuracy of 0.01g +/- 1%

Finding dilution ratio was trial and error. It was obvious that using 100% calibration solution was not an option because the sample was getting too dark. I started with 1:5 but that got me out of range (blinking 200). Then tried 1:10 and this one gave me a reading of 198. My calibration solution was 20ppm NO3 so 198/20=~10. Close enough. That's why the final reading must be divided by 10.

The fact that dilution is 1:10 and reading must be divided by 10 is just a coincidence. Very convenient one It could have been the case that the required dilution was different, e.g. 2.83:10.

Linearity.
I mixed additional calibration solutions (0, 2, 5, 8, 10 and 15ppm) and measured them. Results below:

View attachment 299170

The 2ppm and 15ppm samples are a bit off the perfect line. My calibration solutions were probably a bit off ( I used a syringe, not the accurate scale when diluting 20ppm to 2, 5,8,10 and 15ppm). That's why I consider the test to be 10%+/-1ppm accurate. I could have mixed much better calibration solutions and re-calibrated but 10% accuracy is already more than I need and way better than human eyes + color chart (well, at least my eyes ) The important thing is that results are reproducible.

You can easily calibrate Red Sea test following the same procedure. (EDIT: If other tests give non-linear response, that's not a big deal. We would just need 2-3 more calibration points + an Excel trend line I expect to see linear, logarithmic or exponential responses ).

If you give API a go make sure to warm up the sample and shake it well. My goal was for the reaction to complete in around 3-4min. With cold sample it takes around 7min.

 

[gdemos]

I'd love to hear more on this for NO3 testing accuracy. Any luck using salifert NO3 reagents with Hanna ULR?

 

[doctorwhoreefer]

As much as I want a nitrate meter of some sort, I have to admit I just use the 5 in 1 test strips for a quick and dirty on nitrates.. hehe

I take it the accuracy you've seen so far is indeed fairly repeatable Amebcia?
Have you emailed Hanna customer service with this yet? Maybe a group email to Hanna and API? (that would be cool if they worked together)

 

[amebcia]

I take it the accuracy you've seen so far is indeed fairly repeatable Amebcia?

It is. The key is to make sure the reaction completes fully -> warm up the sample.

Have you emailed Hanna customer service with this yet? Maybe a group email to Hanna and API? (that would be cool if they worked together)

Do not really feel a need to do it. Hoping for few other forum members to confirm my procedure & calibration.

What would Hanna say It is not really in their interest for people to use API reagents. Giving a reliable feedback would need them to do some research, but why would they. Most likely answer is "we cannot comment"

 

[amebcia]

I'd love to hear more on this for NO3 testing accuracy. Any luck using salifert NO3 reagents with Hanna ULR?

Still have plenty of API left so personally I am not planning on calibrating other reagents at this point.

 

[doctorwhoreefer]

I'll have to try this, gotta get some reagents first, but I'll let ya know

 

[amebcia]

I'll have to try this, gotta get some reagents first, but I'll let ya know

Sounds good! Keep me posted.
There is plenty of KNO3 calculators on planted tank forums, but let me know in case you need info on mixing a 20ppm NO3 solution.

 

[msderganc]

Wow, great find!

 

[doctorwhoreefer]

Hanna HI3874 ordered, should see it Wed.

 

[amebcia]

Hanna HI3874 ordered, should see it Wed.

Can't wait for your results This test uses 10ml samples. Same as the ULR Phosphorus. Reaction time is also similar (4min).

One thing that I did not mention before but you may find useful.

To double check that the reaction completed fully (affects accuracy) you can run an extra measurement round once the first one is complete. Just take the original sample (with the color already developed) and take it through both measurement steps again. Zeroing and measurement with 3min timer. Result should show zero.

If you get more than that then we shall mix the sample a bit longer an warm it up.

The need for longer mixing and/or warming up does not mean that original manufacturer instructions are wrong. Both Hanna and API scales are in 10ppm increments while longer mixing and warming up increases the accuracy only by around 10%. Does not make much difference when comparing against a color scale, but an electronic photometer can easily see the difference.

 

[zachfishman]

Hopefully your results are better than mine. I tried using a Hanna Total Chlorine Colorimeter (also uses 525nm light) and Salifert reagents. I figured the Salifert goes from clear to pink/red just like the Hanna Cl- reagents do, whereas the API goes from yellow to red...

I bought multiple nitrate standard solutions (you NEED these, can't calibrate against subjective tests of your tank water), and ran a test with a 30ppm standard... After letting the color develop for 5 minutes I tested, got a reading, yay! I repeated this four more times with the same vial (those Hanna kits have a degree of error), intending to take the average reading and graph it against the NO3 ppm I was testing. But the reading kept increasing every time, and rapidly.

The problem I ran into was that the color never stopped intensifying. With a 30ppm solution, the Hanna might read "0.2" after 5min, "0.6" at 15min, "1.56" at 30min... even at an hour it was still increasing - rising with every successive test less than 1min apart with the same vial!

Hopefully the API or Hanna reagents work better for others. Even doing multiple replicates with 5 different standard solutions between 0 and 150ppm AND waiting an entire hour for the color to plateau (somewhat), I couldn't fit any reasonable best fit line to my scatterplot...

...This might be why Hanna doesn't have a saltwater nitrate colorimeter.

 

[amebcia]

I figured the Salifert goes from clear to pink/red just like the Hanna Cl- reagents do, whereas the API goes from yellow to red...

Does the Salifert kit use 1ml samples? How much of each reagent did you use?

 

[dkeller_nc]

The problem I ran into was that the color never stopped intensifying. With a 30ppm solution, the Hanna might read "0.2" after 5min, "0.6" at 15min, "1.56" at 30min... even at an hour it was still increasing - rising with every successive test less than 1min apart with the same vial.

Something sounds a bit "off". All color-development tests, including nitrate, should be completion reactions such that the most if not all of the nitrate in a given sample is converted to the colored end-product at which point the reaction stops.

Sometimes what can happen in these sorts of analytical wet chemistry assays is that a secondary reaction pathway with an interfering substance is also converting the detection reagent into the colored end-product. If that interfering substance is in great excess, and the secondary reaction pathway is fairly slow, you'd wind up with the behavior you noted.

I'm not sure what that interfering substance might be in your particular situation, but I've set the Salifert nitrate test up in a spectrophotometer a while back, and it does behave the way you expect - fairly rapid color development up to the 2 minute mark, then plateau and stable readings for another 20 minutes.

 

[zachfishman]

Does the Salifert kit use 1ml samples? How much of each reagent did you use?

Yes the Salifert NO3 kit uses 1mL of sample. I used the same amount of reagent (4 drops, 1 scoop) for the 10mL in the Hanna cuvette. I originally doubled the reagents, but my 100ppm nitrate standard solution got too red and exceeded the range of the Hanna checker (after 30min of continued color development). I was hoping to use the kit to test higher levels of nitrate, preferably up to 200ppm within +/-10ppm for FO systems at work.

Although as dkeller_nc noted, I might be experiencing some interference.

 

[zachfishman]

Something sounds a bit "off". All color-development tests, including nitrate, should be completion reactions such that the most if not all of the nitrate in a given sample is converted to the colored end-product at which point the reaction stops.

Sometimes what can happen in these sorts of analytical wet chemistry assays is that a secondary reaction pathway with an interfering substance is also converting the detection reagent into the colored end-product. If that interfering substance is in great excess, and the secondary reaction pathway is fairly slow, you'd wind up with the behavior you noted.

I'm not sure what that interfering substance might be in your particular situation, but I've set the Salifert nitrate test up in a spectrophotometer a while back, and it does behave the way you expect - fairly rapid color development up to the 2 minute mark, then plateau and stable readings for another 20 minutes.

Maybe I need to try this with my own nitrate standard solutions - just RODI and KNO3 powder...

 

[amebcia]

Something sounds a bit "off". All color-development tests, including nitrate, should be completion reactions such that the most if not all of the nitrate in a given sample is converted to the colored end-product at which point the reaction stops.

That would be a reasonable assumption, however zachfishman used standard amount of reagents for 10x the water volume. It is possible that it slows the reaction down.
I believe the reagents that tint a sample pink use the cadmium reduction method and the ones that have yellow/red tint use the nitrazine method.
Details: http://edge.pondev.com/wp-content/uploads/Waste_SM4500-NO3-.pdf

While I do understand the principles and the sequence of the reactions involved, the level of my chemical knowledge does not allow me to predict the minimal amount of regents to be added to get an accurate reading and within a reasonable time.
I am sure RC has plenty of chemistry pros, so maybe somebody will comment

In case of API, when dosing 2x the amount of reagents (because hanna bottle is 10ml not 5ml) reaction stops after 5min (with warming up).

Yes the Salifert NO3 kit uses 1mL of sample. I used the same amount of reagent (4 drops, 1 scoop) for the 10mL in the Hanna cuvette.
I originally doubled the reagents, but my 100ppm nitrate standard solution got too red and exceeded the range of the Hanna checker (after 30min of continued color development).

The reason I am mixing 1ml aquarium water with 9ml RO water is because the hanna P checker is so sensitive that using 100% aquarium water gets me out of range (would probably work when measuring in the <2ppm range).

Did you mix 1ml of the 30ppm solution with 9ml of RO when you added 2x the amount of reagents?

BTW. If we wanted to do it the same way as with API, 10x the amount of Salifert reagents would have to be used. Not quite economical approach and still would need to be proven to work.

Hopefully doctorwhoreefer will give us an update on HI3874. It is designed for 10ml bottles so we are certain what the right amount of reagent is

 

[zachfishman]

Did you mix 1ml of the 30ppm solution with 9ml of RO when you added 2x the amount of reagents?

No dilution, I used 10mL of 30ppm solution. I was hoping that I could use 100% aquarium water without dilution to remove potential variability (when you're dealing with volunteers, all error is possible)

 

[amebcia]

No dilution, I used 10mL of 30ppm solution. I was hoping that I could use 100% aquarium water without dilution to remove potential variability (when you're dealing with volunteers, all error is possible)

Agree. Diluting adds extra error but I do not know a way around it. Accuracy may be improved by using a 1ml syringe.

We must not exceed the range of a colorimeter. In case of Hanna P checker sample at 200ppb P (max) is just slightly blue (when measuring P with the standard Hanna reagents).