Using Hanna Phosphorous Checker (HI-736) To Test for Low Levels of NO3

Rick Mathew

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USING YOUR PHOSPHOROUS HANNA CHECKER HI-736 TO TEST FOR LOW LEVES OF NITRATES

Over the last few weeks I have continued my quest to find a better way to measure low levels of nitrates without having to do color interpretation. Back in October I posted my first efforts. You can find it here: https://www.reef2reef.com/threads/diy-nitrate-test-meter.330892/#post-4114495

From the feedback on this posting I proceeded with the work. Randy provided some insights based on some information from HACH on using a standard salt solution for the calibration (also included in the previous post). Based on my tests the calibration does need to be done using the seawater calibration procedure as seen by Fig 1

FIG 1
upload_2018-1-10_17-17-14.png

clip_image001.gif

clip_image001.gif
clip_image002.gif
clip_image003.gif


This would necessarily mean that the standard solutions would need to be prepared at or near the chloride level of my display tank. That being said I prepared all of my standard solutions in accordingly. I prepared calibration standards at .25ppm, .5ppm, 1ppm and 2ppm using the Hach Nitrate-Nitrogen Standard Solutions (converted from NO3 – N to NO3) I chose these levels because they presented the most difficulty in measuring with my current colorimetric method.

I started the measurements using the Hanna HI-96702 Copper High Range Portable Spectrometer at the .25ppm level. The results were that at this level the instrument was unable to detect the presence of NO3. I tested 5 samples and got mostly “0” or “.01”. It was slightly better at the .5ppm level but the data scatter was too large. So this method would not work….Enter the Phosphorous Checker!!

I noticed when I was doing my monthly check on the Phosphorous checker using the Range Certified Standard Kit that the 100ppb sample was a very light pink…just like the low level (.25,.5ppm) NO3 samples. I wondered if this checker might be able to detect these low levels of Nitrate. I ran 5 tests of the .25ppm using the Phosphorous checker and the results were encouraging (See Table 1)

upload_2018-1-10_17-19-22.png

Table 1
.25ppm NO3 Test Data

With these encouraging results I proceeded to run all 4 levels. I ran 5 separate tests at each level (20 tests) and charted the date. (See Table 2)

upload_2018-1-10_16-42-39.png

Table 2
Meter Readings on HI-736 Checker



Using the data averages I then created a regression equation using Excel that best fit the data. (See Fig 2)

upload_2018-1-10_16-44-2.png

Fig. 2
Regression Equation from Excel



With the correlation coefficient (R2 ) being so high this indicated this might be a winner!. I further refined the equation using a program called MyCurveFit, which is also useful in getting other valuable statistical information that tells you about the quality of the fit and how accurate your predictions might be (F-Test, p-test, and Standard Error of the Estimates) …(Here is a link to the website if you are interested https://www.mycurvefit.com/ ) After refining the equation and looking at the regression analysis data it appears this will work.

Here is the Equation I am using. Y= -.000003258494 x X2 +.01296094 x X +.0534317

Y= the Outcome---The NO3 in PPM X= The HI-736 measurement reading

I am not sure if this equation will work with another HI-736 without creating new calibration curve because I have not tested it.

I have been using this method now for about two months.(See Table 3) I addition I have data from outside testing services (AWT and ATI). I continue to run the NYOS test in parallel with this method. If the level on the NYOS is below 1ppm I have to estimate the value. I also cross check with the Red Sea color wheel to see if I am in the right range…The color differentiation with the Red Sea wheel at the lower levels is still a bit difficult for me to distinguish but it is a good marker to see if my meter readings are in the range.

The procedure is quite simple:

1) Run the Red Sea Nitrate Pro Test according to the procedure.

2) After 7 minutes of the 9 minute reaction time (2minutes remaining) I place a cuvette of the unreacted test water into the Phosphorous Checker and measure this as a blank (C1).

3) I then empty the cuvette and wait for the reaction time to complete.

4) When it is completed I decant off 10mL of the reacted solution and fill the cuvette.

5) I pace the cuvette into the Hanna Phosphorous Checker (HI-736) and press the button (C2). I do not hold the button in to activate the 3 min timer…I just press the button and get the reading.

The short 7 min video below shows the test procedure. The video does not detail the Red Sea Test but only shows the procedure.





Data Results can be seen in Table 3

upload_2018-1-10_16-49-5.png

Table 3
Reference Measurements compared to HI-736 testing results

*These are visually estimated values using NYOS test results and the Red Sea Pro Color Wheel



From my perspective the data looks promising. The errors from the estimated reference measurement are small, most 12% or less with a few (3) exceptions (See Table 3). With my current data set the Standard Error of the Estimate is about .07-.12 depending on the level in PPM. I am going to continue to gather additional data from my own testing as well as outside sources. This will help to refine the regression equation as well as my testing method to increase the accuracy of the prediction. My goal would be to have the Standard Error of the Estimate to be 10% or less of the level tested…. 0.025 for the .25 ppm, 0.05 for the .5ppm etc.

It is important to note that the reference measurements (with the exception of the external tests and knowns) are based on visual assessment using the NYOS color card and the RED SEA PRO Nitrate Wheel. This being the case the value is left to my interpretation. I do the best I can to differentiate between levels that are not specifically on the color chart… EXAMPLE..Looks greater than .25 but less than .5...Looks closer to .5 than .25…must be a .3 or .4 so I take my best guess. I do this first before I do the calculation so as not to influence the value of the reference by the results of the calculation. None the less as you can see the data lines up very well.



I have much more confidence in the known samples that I create for standards and have seen some very good results from these. I only have one outside data point at this time with their value being at .4 ppm and mine at .57 ppm. After reading the posts on R2R on Aqua Medic testing as well as the a Reefs Magazine article by Rich Ross & Dr Maupin on Triton Lab ICP-OES Testing (link here http://packedhead.net/2015/triton-lab-icp-oes-testing-of-a-certified-artificial-saltwater-standard/ ) , I don’t have a high level of confidence that external testing is all that solid of a reference point…In my opinion. I have yet to evaluate the ATI data results and would appreciate any feedback from the community on your experience.

Well that about sums it up. I will continue on my quest and will provide future updates as they are available. For anyone out there who would like to see if they can replicate these results I would be happy to provide any additional details you might need.


FUTURE WORK

· I will also be working on an Iodine test following a similar procedure.

· Refine regression equation for Hanna HI-736 /Red Sea Procedure

· Explore other Hanna Checkers to see what kind of results they produce

· 2 other tests that might be worth exploring…Potassium & Silica



Rick



 
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Ocelaris

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Great work. I always wondered why Hanna doesn't sell a nitrate tester, seems like you're on to something.
 
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Rick Mathew

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Ocelast: 4355565 said:
Great work. I always wondered why Hanna doesn't sell a nitrate tester, seems like you're on to something.

Thank you...appreciate it

Rick
 

Randy Holmes-Farley

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Great work. I always wondered why Hanna doesn't sell a nitrate tester, seems like you're on to something.

It's because of chloride as an interferent, which is why Rick sees a difference between RO/DI and seawater calibration. That makes the results potentially dependent on chloride levels if you do not make your own calibration curve. :)
 
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Rick Mathew

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It's because of chloride as an interferent, which is why Rick sees a difference between RO/DI and seawater calibration. That makes the results potentially dependent on chloride levels if you do not make your own calibration curve. :)

Here is the actual data showing the effects of salinity on the measurement using this test method...

upload_2018-1-11_19-21-11.png
 

Stigigemla

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Is the post (7) relevant for different levels of nitrate. It would be very interesting if it is the same for lets say 50 ppm.
Some of my newbees start nitrate reduction from that level and it would be nice to see if / when the reactors or vodka kick in.
 
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Rick Mathew

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Is the post (7) relevant for different levels of nitrate. It would be very interesting if it is the same for lets say 50 ppm.
Some of my newbees start nitrate reduction from that level and it would be nice to see if / when the reactors or vodka kick in.

Actually the test described in this post is only applicable for NO3 levels 2ppm or less.

The previous work I did might apply to levels as high as 50ppm but I did not test at that level...12ppm is as high as I went. The link at the top of this post will take you to the higher ppm work...Some additional work would need to be done to run calibration curves at these higher levels...The Phosphorous Checker used in this work is only good up to 2ppm above that level with this test the readings are above 200, which is out of the range of the Checker...at least in the work I have done....You would need to explore another measurement instrument, like the one I describe in the link (Hanna Copper HI-96702)...There are other instruments to explore, but I just chose the ones I had and concentrated on the lower end of the concentrations (0-2ppm).... Here is another discussion thread you might be interested in with the same goal in mind

https://www.reef2reef.com/threads/spectrophotometer-question.346685/#post-4319301 Hope this helps

Rick
 
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Rick Mathew

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UPDATE ON USING HI-736 PHOSPHOROUS CHECKER TO MEASURE LOW LEVELS OF NO3


I have continued my efforts on this project and wanted to give an update as to my progress.

First of all I have slightly modified the test procedure to get better repeatability in the measurement; starting with the 1-15-18 measurement. I have added a 2 minute waiting period after I decant off the test solution. This gives better repeatability results. I believe because it allows any particulate matter to settle out and not interfere with the measurement.

I also found a optically matched pair of cuvettes so I can take more than one reading on a single sample. I currently take an avg of 5 readings, but based on consistency of the readings I will be reducing this to 3 or less.

The Test Results Table below shows the current results I have been getting. The errors for the most part are low enough to start putting some confidence in the measurement. The outside testing service errors are still large (23%) but better than the origional (42%). As I mentioned before, I do not have a high degree of confidnce in the results of the outside testing services on testing NO3, but I included them as data points in the analysis. The results of the known samples (Highlighted in yellow) are still the ones I have the most confidence in.

YELLOW HIGHLIGHT = KNOWN PREPARED IN SALTWATER

GREEN HIGHLIGHT = OUTSIDE TESTING SERVICES RESULTS

NO HIGHLIGHT= ACTUAL SAMPLE MEASUREMENTS FROM MY TANK


upload_2018-2-2_11-44-10.png


TEST DATA RESULTS AS OF 2-1-18

 

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The absorption peak for the nitrate kits is 540nm verified. The peak is super wide 520-570. Any old green led in this range will work without a filter so long as you follow the above calibration when making standards. How long do you need to let the seawater sit before you make up your standards? For example, I want to test my tank so I measure salinity and then make up some seawater from my salt mix. I use this as a zero blank but also to mix up the standards for the abs points. How long does the water need to stand? I figure float it in the tank for temp correction.
 
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Rick Mathew

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The absorption peak for the nitrate kits is 540nm verified. The peak is super wide 520-570. Any old green led in this range will work without a filter so long as you follow the above calibration when making standards. How long do you need to let the seawater sit before you make up your standards? For example, I want to test my tank so I measure salinity and then make up some seawater from my salt mix. I use this as a zero blank but also to mix up the standards for the abs points. How long does the water need to stand? I figure float it in the tank for temp correction.


I mix mine up and let it set for 24 hours...but I an not sure that is necessary. I use reagent grade Sodium Chloride to make up my calibration water and target 3.29% by weight...
 

kecked

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Thanks/. I have a deuterium light coming. I will compare this test to 200-210nm peak absorption. I also want to try adding lead to make lead chloride. It’s insoluble. Just curious if I remove the chloride what would happen. Not an environmental test for sure but we are using cadmium already. Lead acetate is soluble and should drop the chloride out as a yellow precip leaving acetic acid/Na behind. Changes the pH though.

Nice paper
https://www.sciencedirect.com/science/article/pii/S0304420305001714
 
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Rick Mathew

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Thanks/. I have a deuterium light coming. I will compare this test to 200-210nm peak absorption. I also want to try adding lead to make lead chloride. It’s insoluble. Just curious if I remove the chloride what would happen. Not an environmental test for sure but we are using cadmium already. Lead acetate is soluble and should drop the chloride out as a yellow precip leaving acetic acid/Na behind. Changes the pH though.

Nice paper
https://www.sciencedirect.com/science/article/pii/S0304420305001714

Interesting approach...this paper you found is very interesting...I am going to have a look...

Thanks

rick
 

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I found a second one using resorcinol. There are a lot of ways to test for nitrate in sea water. We should not be limited to the test kits available. I really want to get rid of the cadmium. IF this can be done purely as spec it would be best. There are now uv leds though they are not yet in the range I want 200-210nm
 

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Ok got spec and light setup. Ran the red sea nitrate test and just looked at the result. Full development takes 4 minutes. Stay stable so far for an hour. Peak absorbance 550nm. I see no reason I can’t just do standards for this test and run the result.

So whatever unit they have set for 520-570 nm should work ok. 540-560 preferred. The peak is very wide.

Someone wanted. Me compare the reflectancebof the card and the actual solution. The peak is around 650 and same for the solution. It’s pretty close. I was surprised.
 
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USING YOUR PHOSPHOROUS HANNA CHECKER HI-736 TO TEST FOR LOW LEVES OF NITRATES

Over the last few weeks I have continued my quest to find a better way to measure low levels of nitrates without having to do color interpretation. Back in October I posted my first efforts. You can find it here:
https://www.reef2reef.com/threads/diy-nitrate-test-meter.330892/#post-4114495

From the feedback on this posting I proceeded with the work. Randy provided some insights based on some information from HACH on using a standard salt solution for the calibration (also included in the previous post). Based on my tests the calibration does need to be done using the seawater calibration procedure as seen by Fig 1

FIG 1
upload_2018-1-10_17-17-14.png

clip_image001.gif

clip_image001.gif
clip_image002.gif
clip_image003.gif


This would necessarily mean that the standard solutions would need to be prepared at or near the chloride level of my display tank. That being said I prepared all of my standard solutions in accordingly. I prepared calibration standards at .25ppm, .5ppm, 1ppm and 2ppm using the Hach Nitrate-Nitrogen Standard Solutions (converted from NO3 – N to NO3) I chose these levels because they presented the most difficulty in measuring with my current colorimetric method.

I started the measurements using the Hanna HI-96702 Copper High Range Portable Spectrometer at the .25ppm level. The results were that at this level the instrument was unable to detect the presence of NO3. I tested 5 samples and got mostly “0” or “.01”. It was slightly better at the .5ppm level but the data scatter was too large. So this method would not work….Enter the Phosphorous Checker!!

I noticed when I was doing my monthly check on the Phosphorous checker using the Range Certified Standard Kit that the 100ppb sample was a very light pink…just like the low level (.25,.5ppm) NO3 samples. I wondered if this checker might be able to detect these low levels of Nitrate. I ran 5 tests of the .25ppm using the Phosphorous checker and the results were encouraging (See Table 1)

upload_2018-1-10_17-19-22.png

Table 1
.25ppm NO3 Test Data

With these encouraging results I proceeded to run all 4 levels. I ran 5 separate tests at each level (20 tests) and charted the date. (See Table 2)


upload_2018-1-10_16-42-39.png

Table 2
Meter Readings on HI-736 Checker


Using the data averages I then created a regression equation using Excel that best fit the data. (See Fig 2)

upload_2018-1-10_16-44-2.png

Fig. 2
Regression Equation from Excel


With the correlation coefficient (R2 ) being so high this indicated this might be a winner!. I further refined the equation using a program called MyCurveFit, which is also useful in getting other valuable statistical information that tells you about the quality of the fit and how accurate your predictions might be (F-Test, p-test, and Standard Error of the Estimates) …(Here is a link to the website if you are interested https://www.mycurvefit.com/ ) After refining the equation and looking at the regression analysis data it appears this will work.

Here is the Equation I am using. Y= -.000003258494 x X2 +.01296094 x X +.0534317

Y= the Outcome---The NO3 in PPM X= The HI-736 measurement reading

I am not sure if this equation will work with another HI-736 without creating new calibration curve because I have not tested it.

I have been using this method now for about two months.(See Table 3) I addition I have data from outside testing services (AWT and ATI). I continue to run the NYOS test in parallel with this method. If the level on the NYOS is below 1ppm I have to estimate the value. I also cross check with the Red Sea color wheel to see if I am in the right range…The color differentiation with the Red Sea wheel at the lower levels is still a bit difficult for me to distinguish but it is a good marker to see if my meter readings are in the range.

The procedure is quite simple:

1) Run the Red Sea Nitrate Pro Test according to the procedure.

2) After 7 minutes of the 9 minute reaction time (2minutes remaining) I place a cuvette of the unreacted test water into the Phosphorous Checker and measure this as a blank (C1).

3) I then empty the cuvette and wait for the reaction time to complete.

4) When it is completed I decant off 10mL of the reacted solution and fill the cuvette.

5) I pace the cuvette into the Hanna Phosphorous Checker (HI-736) and press the button (C2). I do not hold the button in to activate the 3 min timer…I just press the button and get the reading.


The short 7 min video below shows the test procedure. The video does not detail the Red Sea Test but only shows the procedure.




Data Results can be seen in Table 3

upload_2018-1-10_16-49-5.png

Table 3
Reference Measurements compared to HI-736 testing results

*These are visually estimated values using NYOS test results and the Red Sea Pro Color Wheel


From my perspective the data looks promising. The errors from the estimated reference measurement are small, most 12% or less with a few (3) exceptions (See Table 3). With my current data set the Standard Error of the Estimate is about .07-.12 depending on the level in PPM. I am going to continue to gather additional data from my own testing as well as outside sources. This will help to refine the regression equation as well as my testing method to increase the accuracy of the prediction. My goal would be to have the Standard Error of the Estimate to be 10% or less of the level tested…. 0.025 for the .25 ppm, 0.05 for the .5ppm etc.

It is important to note that the reference measurements (with the exception of the external tests and knowns) are based on visual assessment using the NYOS color card and the RED SEA PRO Nitrate Wheel. This being the case the value is left to my interpretation. I do the best I can to differentiate between levels that are not specifically on the color chart… EXAMPLE..Looks greater than .25 but less than .5...Looks closer to .5 than .25…must be a .3 or .4 so I take my best guess. I do this first before I do the calculation so as not to influence the value of the reference by the results of the calculation. None the less as you can see the data lines up very well.



I have much more confidence in the known samples that I create for standards and have seen some very good results from these. I only have one outside data point at this time with their value being at .4 ppm and mine at .57 ppm. After reading the posts on R2R on Aqua Medic testing as well as the a Reefs Magazine article by Rich Ross & Dr Maupin on Triton Lab ICP-OES Testing (link here
http://packedhead.net/2015/triton-lab-icp-oes-testing-of-a-certified-artificial-saltwater-standard/ ) , I don’t have a high level of confidence that external testing is all that solid of a reference point…In my opinion. I have yet to evaluate the ATI data results and would appreciate any feedback from the community on your experience.

Well that about sums it up. I will continue on my quest and will provide future updates as they are available. For anyone out there who would like to see if they can replicate these results I would be happy to provide any additional details you might need.


FUTURE WORK


· I will also be working on an Iodine test following a similar procedure.

· Refine regression equation for Hanna HI-736 /Red Sea Procedure

· Explore other Hanna Checkers to see what kind of results they produce

· 2 other tests that might be worth exploring…Potassium & Silica



Rick





@Psup thought this might interest you since you hate color charts
 

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