Stability Of Hanna Alkalinity Reagent

Dan_P

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We have reports that alkalinity measurements made with reagent taken from a previously used bottle of the Hanna alkalinity reagent differ from the measurement obtained with reagent taken from a newly opened bottle. Also reported is that this discrepancy is eliminated when the reagent is refrigerated between uses. I was unable to reproduce this effect with an eight month stability test. Before getting any deeper into this discrepancy, I performed a series of stress tests on a newly open bottle of reagent in an attempt to make the reagent misbehave. I looked at how evaporation, overcharges, and heat affected alkalinity measurements. The data is presented below.

The first measurement is the control. The reagent from a newly opened reagent bottle was used to messure the alkalinity of Instant Ocean. From this measurement I calculated Hanna’s expected range in value for the measurement and plotted it as two red lines on the plot. The next test was just putting 1.5 mL of reagent in the evaporating dish and then immediately using it. This shows that moving the sample in snd out of the evaporating dish does not change the reagent. The subsequent samples with increasing amount of percent evaporation seemed to show a an upward trend in alkalinity. Only at 19% evaporation do I feel comfortable saying that I am not observing noise in the data (this seems to say that the Hanna Checker measurement is insensitive to reagent evaporation).

I also looked at the effect of screwing up the reagent addition. In the first screw up I added an extra 0.1 mL of reagent (slight effect) and the second I really screwed things up by undercharging the sample by 0.5 mL and adding an extra 0.5 mL of reagent. In the latter case I saw a strong effect, but not necessarily large enough to notice during a routine measurement, maybe.

In the last test, I placed the reagent in boiling water for 10 minutes. After cooling the sample to room temperature and confirming no evaporation occurred, I performed an alkalinity measurement. There was no effect.

Question: which real life variable(s) are my stability and stress studies missing that exist in the real life situations where aquarists observe reagent instability?


image.png
 

Randy Holmes-Farley

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An unknown difference between different folks experiences may relate to differences in quality control aspects such as purity of the dyes present.

Absorbance of a single dye, of course, is concentration related, but a different dye present as an impurity may have far different (potentially higher) absorbance. That is why products can yellow over time without a large portion of the material degrading, for example.
 

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I question if the reagent is a homogenous mixture and stays that way while stored. I swirl the bottle around before every test and haven’t had an issue. The longest I have had a bottle previously opened and stored is about 3 months, and I estimate that I have used about 20 bottles of reagent since I’ve owned the HI772 checker. Even down to the last test, it is within the margin of error when compared to my other tester. But it could be coincidental as well.
 
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An unknown difference between different folks experiences may relate to differences in quality control aspects such as purity of the dyes present.

Absorbance of a single dye, of course, is concentration related, but a different dye present as an impurity may have far different (potentially higher) absorbance. That is why products can yellow over time without a large portion of the material degrading, for example.

To study this possibility, I would need folks to send me their old bottles of reagent they believe to have acted oddly. I could then look for such differences.
 
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Dan_P

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My r
I question if the reagent is a homogenous mixture and stays that way while stored. I swirl the bottle around before every test and haven’t had an issue. The longest I have had a bottle previously opened and stored is about 3 months, and I estimate that I have used about 20 bottles of reagent since I’ve owned the HI772 checker. Even down to the last test, it is within the margin of error when compared to my other tester. But it could be coincidental as well.
OK, swirling will go on the list of possible hints about what is going on,
 
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An unknown difference between different folks experiences may relate to differences in quality control aspects such as purity of the dyes present.

Absorbance of a single dye, of course, is concentration related, but a different dye present as an impurity may have far different (potentially higher) absorbance. That is why products can yellow over time without a large portion of the material degrading, for example.
One more thought on this notion. I think the reagent fails low not high. Users are ending up measuring higher alkalinity with newly opened reagent bottle. That’s my impression anyway.
 

Randy Holmes-Farley

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One more thought on this notion. I think the reagent fails low not high. Users are ending up measuring higher alkalinity with newly opened reagent bottle. That’s my impression anyway.

Some companies manufacture products for the EU in the EU for various reasons. Maybe different users get products from different plants.

Do yours say where it is made? Maybe Lasse’s is from a different plant.
 
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Some companies manufacture products for the EU in the EU for various reasons. Maybe different users get products from different plants.

Do yours say where it is made? Maybe Lasse’s is from a different plant.
I will look when the new bottles arrive. I seem to recall that many products from Hanna are made in Romania.
 

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Hopefully this is another data point to help solve this interesting puzzle.

I always calibrate each bottle of Hanna alkalinity reagent to a commercial alkalinity standard (just for the fun of it), and absolutely saw this drift over time as the reagent is consumed.

My thinking at the time was as the reagent is consumed, a larger and larger volume of the reagent bottle contains air, and while the mechanism is unknown, the summation of up to 25 cycles of fresh air over time induces the drift. I think at the time I was testing one tank every couple or few days, so the bottle would last around 2 months. By the end of two months or so, there would typically be significant drift. Looking back at the log, here are some of the jumps I recorded:

8.4 to 9.1
8.5 to 9.6
7.2 to 7.5

My solution is to open a new container and immediately transfer the reagent to three 10ml glass vials to reduce the number of open/close cycles, and the total amount of air the reagent is exposed to. Been doing this for years, and it seems to work, zero drift. No refrigeration used, but i really like the refrigeration idea.

Vials.png


It’s common to see floating slime or bacteria in new reagent. I wonder if some type of biological activity is responsible for the drift.
 

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since i no longer remove the seal cap and use a syringe with a needle to take the 1ml.
i have the impression that it is more stable...
the shift seems smaller. (Gap between old and new bottle)
No refrigeration ...
but i didn't document it...
1000004884.jpg
 

areefer01

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Probably already know or thought of:

  • Didn't Lasse do a series of Hanna's Alk and refrigeration?
  • Age of battery new vs uses vs old - not sure if it matters?
  • Lot number?
  • Test vial/cuvette?
  • Instructions for Alk -

Not the same I understand but my Hanna Nitrate checker was inconsistent between tests. I replaced the batter and that took care of it. About a month later results started to vary again. Thought it was batter but this time after replacing it the same behavior occurred. I looked at the reagent box, lot number, expiration date and it was good until 2025. I had an unopened box, different lot number, good expiration date, and numbers back to consistent numbers after tests. I was testing daily at the same time.

Test vial, cuvette, and cap. Again back to the Nitrate and my daily testing I started to see the results vary day by day. Couldn't figure out why so looked at what I was doing. I have several Hanna cuvettes from Alk, Phosphate, and Nitrate testers and put them in a drawer. Grab one, do the test, rinse later, put back in drawer. Next day I made sure to use the same cuvette and the number was consistent. Marked that one and that is the only one I use now.

Alk I found was more sensitive to shaking and would cause higher readings. I would swirl and gently flip upside down once or twice and then take the measurement. The quicker the better.

All of this you already know so was just spit balling. I've had odd luck with the Nitrate but Alk has been ok. I'm not looking for exact though so maybe my expectations are different.

Hope your evening is well.
 

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since i no longer remove the seal cap and use a syringe with a needle to take the 1ml.
i have the impression that it is more stable...
the shift seems smaller. (Gap between old and new bottle)
No refrigeration ...
but i didn't document it...
1000004884.jpg
Neat idea. Brownie points. :) I’m going try that for all my Hanna reagents.

A bit off topic and I know Randy doesn’t like MG testing but one thing I find is the MG reagent bottle gets lots of precipitation on it even when stored tightly and upright so clearly the lids seals on these reagents is less than perfect so I can see this potentially helping on all the liquid reagents. MG reagent makes it very evident so I can see the same correlation to the other bottles as the lids are seemingly the same
 
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Dan_P

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By the end of two months or so, there would typically be significant drift. Looking back at the log, here are some of the jumps I recorded:

8.4 to 9.1
8.5 to 9.6
7.2 to 7.5
Just to clarify, from the first example above, 8.4 is the measurement of the standard when the bottle was newly opened and 9.1 is an alkalinity measurement after two months or when the bottle was nearly empty?
 

RelaxingWithTheReef

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Just to clarify, from the first example above, 8.4 is the measurement of the standard when the bottle was newly opened and 9.1 is an alkalinity measurement after two months or when the bottle was nearly empty?
No, I should of been clearer. 8.4 is the last test of the old two month bottle with tank water, and 9.1 is the reading from a new (calibrated) bottle with the same tank.

Each reagent bottle is initially calibrated to an alkalinity standard, and a correction noted to eliminate the normal batch to batch variation.

BTW - Been doing the calibration for years, and so far the correction is always within +- 0.2ppm, and within the Hanna published accuracy.

All of these test pairs were taken together at the the same time with the same tank water for the purpose of measuring continuity between bottles of reagents. I knew there was a drift problem at the time, and was trying to understand it better.
 
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No, I should of been clearer. 8.4 is the last test of the old two month bottle with tank water, and 9.1 is the reading from a new (calibrated) bottle with the same tank.

Each reagent bottle is initially calibrated to an alkalinity standard, and a correction noted to eliminate the normal batch to batch variation.

BTW - Been doing the calibration for years, and so far the correction is always within +- 0.2ppm, and within the Hanna published accuracy.

All of these test pairs were taken together at the the same time with the same tank water for the purpose of measuring continuity between bottles of reagents. I knew there was a drift problem at the time, and was trying to understand it better.
Thank you for clarifying. You’ve done a thorough study and your reagent storage method gives me a lead to study.
 
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Dan_P

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since i no longer remove the seal cap and use a syringe with a needle to take the 1ml.
i have the impression that it is more stable...
the shift seems smaller. (Gap between old and new bottle)
No refrigeration ...
but i didn't document it...
1000004884.jpg
Oh, this is clever. Thanks.
 
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Probably already know or thought of:

  • Didn't Lasse do a series of Hanna's Alk and refrigeration?
  • Age of battery new vs uses vs old - not sure if it matters?
  • Lot number?
  • Test vial/cuvette?
  • Instructions for Alk -

Not the same I understand but my Hanna Nitrate checker was inconsistent between tests. I replaced the batter and that took care of it. About a month later results started to vary again. Thought it was batter but this time after replacing it the same behavior occurred. I looked at the reagent box, lot number, expiration date and it was good until 2025. I had an unopened box, different lot number, good expiration date, and numbers back to consistent numbers after tests. I was testing daily at the same time.

Test vial, cuvette, and cap. Again back to the Nitrate and my daily testing I started to see the results vary day by day. Couldn't figure out why so looked at what I was doing. I have several Hanna cuvettes from Alk, Phosphate, and Nitrate testers and put them in a drawer. Grab one, do the test, rinse later, put back in drawer. Next day I made sure to use the same cuvette and the number was consistent. Marked that one and that is the only one I use now.
If you are referring to the high range nitrate Checker, I too found a devoted vial minimized variation. I think it is related to the small amount of nitrate reducing agent used in the reagent formulation.

Alk I found was more sensitive to shaking and would cause higher readings. I would swirl and gently flip upside down once or twice and then take the measurement. The quicker the better.
Definitely need to look into this and see if I can reproduce the effect.
 

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Hopefully this is another data point to help solve this interesting puzzle.

I always calibrate each bottle of Hanna alkalinity reagent to a commercial alkalinity standard (just for the fun of it), and absolutely saw this drift over time as the reagent is consumed.

My thinking at the time was as the reagent is consumed, a larger and larger volume of the reagent bottle contains air, and while the mechanism is unknown, the summation of up to 25 cycles of fresh air over time induces the drift. I think at the time I was testing one tank every couple or few days, so the bottle would last around 2 months. By the end of two months or so, there would typically be significant drift. Looking back at the log, here are some of the jumps I recorded:

8.4 to 9.1
8.5 to 9.6
7.2 to 7.5

My solution is to open a new container and immediately transfer the reagent to three 10ml glass vials to reduce the number of open/close cycles, and the total amount of air the reagent is exposed to. Been doing this for years, and it seems to work, zero drift. No refrigeration used, but i really like the refrigeration idea.

Vials.png


It’s common to see floating slime or bacteria in new reagent. I wonder if some type of biological activity is responsible for the drift.
Might be unrelated but I used to use Red Sea alkalinity pro kit, and I saw the floating slime/particles in the reagent of a new test kit. I contacted them and sent pictures and they said the reagent is bad and they sent me two replacement bottles.

What is this stuff? Does it mean the reagent will give false results?
 

Randy Holmes-Farley

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Might be unrelated but I used to use Red Sea alkalinity pro kit, and I saw the floating slime/particles in the reagent of a new test kit. I contacted them and sent pictures and they said the reagent is bad and they sent me two replacement bottles.

What is this stuff? Does it mean the reagent will give false results?

While floaters in Hanna tests are often an issue, including simple dust, they wouldn’t always do so in other color change tests unless they actually impacted certain chemical concentrations, and without knowing what it is, one cannot know the effect.
 
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Dan_P

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I wanted to thank you all for your contributions to this post. Here is how I sum things up.

Most of the reefing world has observed the Hanna alkalinity reagent degrade after opening a new bottle. The degradation is obvious after as little time as two months. The degradation manifests itself as an underestimation of the alkalinity, i.e., the measurement is lower than when the reagent was fresh. Refrigerating the sample or scrupulously excluding air minimizes or eliminates the effect. On the other hand, my alkalinity reagent was stable for eight months and when I attempted to stress the reagent through evaporation, heating or overcharging, the measured alkalinity was an overestimate, i.e., the measurements were higher than the control.

Back to the drawing board, making the alkalinity reagent fail..
 

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