Measuring Organic Matter. A Brief Study

[Dan_P]

Hey Dan,

I have prepared freshly mixed 35ppt water, the 390ppm KMnO4 solution, and the 2% NaOH solution. I can also sterile filter tank water and skimmate of course.

What other dilutions for the KMnO4 solution do you recommend I should try?

Your instructions say to use 5mL sample, 1mL NaOH solution and 1mL KMnO4 solution. The sample vials require 10mL but I assume 7mL will be enough to reach the height of the photosensor in the HannaChecker.

I assume you are zeroing the HannaChecker with the same water used for the sample?

The spacer height is 1.7 cm.

For some reason, 1.7 cm is the maximum height limit. Above this height, the Checker response starts to change. Never bothered to discover why.

 

[EnterName]

Oh sorry, one important detail I left out for using the Checkers for measuring a 5 mL (or 7 mL) solutions. You need to put spacer under the vial to raise it up to the light path. Since the lid won’t close you will need to cover the protruding vial. I use an opaque pill bottle but aluminum foil will work or anything that will prevent stray from light entering the Checker.

As for the spacer, i think it’s 1.7 cm but I will confirm that tomorrow. I cut the rounded end of a cylindrical pipette bulb to size. Luckily this was the correct diameter to easily fit into the Check and securely support the vial. There must be hundreds of things that can serve as a spacer.

Hey Dan, I just experimented a bit (full protocol will be posted once I double checked):

Creating the 2% NaOH and 390ppm KMnO4 solution was easy enough. I additionally used a 195ppm KMnO4 and a 292.5ppm KMnO4 solution (so 50% and 75% of the original 390ppm). The color changed from the typical violet to an orange/salmon while the 6 vials were boiling (3 prepared with freshly mixed saltwater and 3 with aquarium water). The precipitate amount varied between freshly mixed saltwater and aquarium water depending on the used KMnO4 solution by 10 - 50%. So far the experiment seemed to be going well.

However: After centrifuging, decanting, and filtering the solutions to fully get rid of any residual precipitate, all solutions came out crystal clear and the Nitrate LR HannaChecker did not manage to detect differences between the freshly mixed water/aquarium water, and the prepared vial samples. I will try with skimmate water next to see if there will be measurable results and decided to prepare twice the amount, so I don't even have to bother with lifting the vial and ensuring no light can come in through the top.

The sterile filtering really took the last bit of coloration out of the solutions, but without it, I would have introduced residues of the precipitate into the test vial. I will also try different vials that will allow for more pressure to build up during the boiling step. The ones I used probably weren't air tight enough to allow for enough pressure.

 

[Randy Holmes-Farley]

The spacer height is 1.7 cm.

For some reason, 1.7 cm is the maximum height limit. Above this height, the Checker response starts to change. Never bothered to discover why.

Probably the light beam gets blocked too much by the spacer, if I am understanding correctly.

 

[Dan_P]

Probably the light beam gets blocked too much by the spacer, if I am understanding correctly.

The spacer is below the vial. Here is more info.

Picture the light beam passing through the vial. Taricha showed that the light beam is not a simply cylinder of light because it passes through a convex then a concave surface before hitting the detector. Anyway as you move the vial upwards, the Checker doesn’t “notice” until the vial is 1.7 cm off the bottom of the Checker. At that point it starts detecting the optical density differently and no longer reads the standard correctly. This a repeatable phenomenon with any vial. I never pursued this further.

 

[Randy Holmes-Farley]

The spacer is below the vial. Here is more info.

Picture the light beam passing through the vial. Taricha showed that the light beam is not a simply cylinder of light because it passes through a convex then a concave surface before hitting the detector. Anyway as you move the vial upwards, the Checker doesn’t “notice” until the vial is 1.7 cm off the bottom of the Checker. At that point it starts detecting the optical density differently and no longer reads the standard correctly. This a repeatable phenomenon with any vial. I never pursued this further.

Right. If the beam extends down to 1.5 cm above the bottom of the chamber, then a 1.7 cm spacer will begin to block it.

 

[Dan_P]

Hey Dan, I just experimented a bit (full protocol will be posted once I double checked):

Creating the 2% NaOH and 390ppm KMnO4 solution was easy enough. I additionally used a 195ppm KMnO4 and a 292.5ppm KMnO4 solution (so 50% and 75% of the original 390ppm). The color changed from the typical violet to an orange/salmon while the 6 vials were boiling (3 prepared with freshly mixed saltwater and 3 with aquarium water). The precipitate amount varied between freshly mixed saltwater and aquarium water depending on the used KMnO4 solution by 10 - 50%. So far the experiment seemed to be going well.

However: After centrifuging, decanting, and filtering the solutions to fully get rid of any residual precipitate, all solutions came out crystal clear and the Nitrate LR HannaChecker did not manage to detect differences between the freshly mixed water/aquarium water, and the prepared vial samples. I will try with skimmate water next to see if there will be measurable results and decided to prepare twice the amount, so I don't even have to bother with lifting the vial and ensuring no light can come in through the top.

The sterile filtering really took the last bit of coloration out of the solutions, but without it, I would have introduced residues of the precipitate into the test vial. I will also try different vials that will allow for more pressure to build up during the boiling step. The ones I used probably weren't air tight enough to allow for enough pressure.

The color intensity of permanganate in freshly mixed saltwater shouldn’t change much after heating. I use Instant Ocean. Is there organic compounds in your salt mix? Or a level high level of trace elements?

If the test solution was pink to purple after heating but was colorless after filtration, that points to the permanganate oxidizing the filter medium. The Checker can be forgiving of floating particles. Cloudiness not so much.

Full strength skimmate can completely decolorize the permanganate solution at room temperature. You will need to dilute it.

Keeping the caps loose during heating should not result in much evaporation or affect the course of the reaction.

 

[EnterName]

The color intensity of permanganate in freshly mixed saltwater shouldn’t change much after heating. I use Instant Ocean. Is there organic compounds in your salt mix? Or a level high level of trace elements?

If the test solution was pink to purple after heating but was colorless after filtration, that points to the permanganate oxidizing the filter medium. The Checker can be forgiving of floating particles. Cloudiness not so much.

Full strength skimmate can completely decolorize the permanganate solution at room temperature. You will need to dilute it.

Keeping the caps loose during heating should not result in much evaporation or affect the course of the reaction.

I used Nyos pure which does not contain organic additives nor excessive trace element amounts. You can find the ICP-MS results of the batch I'm using here.

The color fully disappeared during the boiling process regardless of using freshly mixed salt water, tank water, or undiluted skimmate water (which was amber colored initially). The color was therefore gone before the KMnO₄ came in contact with any filter material.

I will retry the experiment with a second batch of freshly prepared salt water, and maybe 1:10 diluted tank water or so. I think using RO/DI for the dilution might be the safer bet just in case for some reason the salt batch does indeed contain any organics? I will avoid using stainless steel syringe tips just in case it makes a difference, but usually 0.01mol or lower shouldn't be agressive enough to attack the stainless steel significantly.

How I prepared the required stock solutions: (just in case my math is off)
The NaOH solution was prepared using 10g NaOH (> 99%, food grade) and approx. 500mL RO/DI (0.053µS/cm at 15°C/59°F). I dissolved the 10g NaOH in 450mL water and filled up to the 500mL mark once it reached 20°C/68°F. I used a 500mL measuring cylinder to be as precise as possible.

I only had access to a 0.01mol/L KMnO₄ standard solution. This means it is 1.5803g/L or 1580.3ppm

(0.01mol/L ⋅ 158.03g/mol KMnO₄ = 1.5803g/L KMnO₄ = 1580.3mg/L KMnO₄)​

To create 1L of a 390ppm KMnO₄ solution (containing 390mg in total) I would need 24.7% of the 0.01mol solution.

(390mg / 1580.3mg ≈ 0.247 = 24.7%)​

I didn't want to waste 1/4 of the stock solution already, so I used 2.47% (24.7mL) and diluted it up to 100mL with RO/DI instead of 1L.

 

[Dan_P]

I used Nyos pure which does not contain organic additives nor excessive trace element amounts. You can find the ICP-MS results of the batch I'm using here.

The color fully disappeared during the boiling process regardless of using freshly mixed salt water, tank water, or undiluted skimmate water (which was amber colored initially). The color was therefore gone before the KMnO₄ came in contact with any filter material.

I will retry the experiment with a second batch of freshly prepared salt water, and maybe 1:10 diluted tank water or so. I think using RO/DI for the dilution might be the safer bet just in case for some reason the salt batch does indeed contain any organics? I will avoid using stainless steel syringe tips just in case it makes a difference, but usually 0.01mol or lower shouldn't be agressive enough to attack the stainless steel significantly.

How I prepared the required stock solutions: (just in case my math is off)
The NaOH solution was prepared using 10g NaOH (> 99%, food grade) and approx. 500mL RO/DI (0.053µS/cm at 15°C/59°F). I dissolved the 10g NaOH in 450mL water and filled up to the 500mL mark once it reached 20°C/68°F. I used a 500mL measuring cylinder to be as precise as possible.

I only had access to a 0.01mol/L KMnO₄ standard solution. This means it is 1.5803g/L or 1580.3ppm

(0.01mol/L ⋅ 158.03g/mol KMnO₄ = 1.5803g/L KMnO₄ = 1580.3mg/L KMnO₄)​

To create 1L of a 390ppm KMnO₄ solution (containing 390mg in total) I would need 24.7% of the 0.01mol solution.

(390mg / 1580.3mg ≈ 0.247 = 24.7%)​

I didn't want to waste 1/4 of the stock solution already, so I used 2.47% (24.7mL) and diluted it up to 100mL with RO/DI instead of 1L.

Reagent preparation looks correct.

The loss of color is a mystery. One last check is to skip the heating step, work up the sample and measure the color intensity. You can do this in RO/DI to avoid the precipitate and skip the work up.

 

[EnterName]

Reagent preparation looks correct.

The loss of color is a mystery. One last check is to skip the heating step, work up the sample and measure the color intensity. You can do this in RO/DI to avoid the precipitate and skip the work up.

A = 10mL RO/DI + 2mL 2% NaOH + 2mL 390ppm KMnO₄
B = 10mL 35ppt Nyos Pure Saltwater + 2mL 2% NaOH + 2mL 390ppm KMnO₄
C = 10mL 35ppt Tank water + 2mL 2% NaOH + 2mL 390ppm KMnO₄

The vials are not heated yet (currently at 20°C/68°F). I will let the vials sit for a few minutes and check if the color disappears without the heating step. If not, I will try heating again to see what happens to the RO/DI compared to the other two vials.

I will also add a vial D with 1mL tank water, 9mL RO/DI + 2mL 2% NaOH + 2mL 390ppm KMnO₄.

Note: I doubled the reagent this time so I don't have to rely on a spacer for the HannaChecker. In my first run I followed the 5mL + 1mL + 1mL instructions in case double the reagents would somehow cause issues.

 

[Dan_P]

A = 10mL RO/DI + 2mL 2% NaOH + 2mL 390ppm KMnO₄
B = 10mL 35ppt Nyos Pure Saltwater + 2mL 2% NaOH + 2mL 390ppm KMnO₄
C = 10mL 35ppt Tank water + 2mL 2% NaOH + 2mL 390ppm KMnO₄

The vials are not heated yet (currently at 20°C/68°F). I will let the vials sit for a few minutes and check if the color disappears without the heating step. If not, I will try heating again to see what happens to the RO/DI compared to the other two vials.

I will also add a vial D with 1mL tank water, 9mL RO/DI + 2mL 2% NaOH + 2mL 390ppm KMnO₄.

Are you working late tonight? :-)

 

[EnterName]

Are you working late tonight? :-)

It's 9:30pm here, I'm comfortably at home and enjoy doing some chemistry in my "home-lab". Nothing about this feels like work to me :)

I'm a computer scientist/software dev so this really is just something I love to learn and do for fun :)

 

[Christoph]

Hello,

interesting, looking forward to your results!

What needs to be kept in mind is that Permanganate is not a very strong oxidizer - so not all organics will react. Very reactive is everything thats unsaturated.

What might be worth considering is switching from basic to acidic reaction conditions: Thus the reaction product should be likely Mn2+ ions instead of insoluble brown MnO4 - in addition the reaction mixture would likely be homogenous (the hydroxide precipitates magnesium salts), which might add some benefit in downstream photometry. But maybe there are also drawbacks, especially in seawater.

Edit: After thinking about it a bit longer it might not be a good idea, because Chlorine might be formed. If you want to try it, do it only at the microscale!

BR, Christoph

 

[EnterName]

Okay, this time the colors remained for all 4 samples.

I did not try a pure RO/DI sample before, so A is "new" to me. I prepared B and C just like before (just doubled the sample size and reagents). The only other difference is that I didn't use a syringe filter this time before adding NaOH and KMnO₄. Originally I used a Nylon 0.22µm filter. Maybe it released something into the samples which later on reacted with the KMnO₄ once I added the reagents?

I'm not yet sure if the colors make sense though.

• A is pure RO/DI and should have kept its color pretty well if I understand correctly what is happening. Maybe I grabbed the wrong canister and used RO/DI that has been sitting openly for a day.​
• B is freshly mixed 35ppt saltwater and I understand that it kept its color quite well (I would have expected A to look similar).​
• C is tank water and probably full of organics, so it is fair that it lost some of its color.​
• D is 1mL tank water and 9mL RO/DI and should be between A and C, but instead is more like B and C.​

Readings (zeroed with 35ppt freshly mixed saltwater):

• A: 1.44
• B: 1.70
• C: 0.75
• D: 1.19

Absolutely no syringe filters were used.

 

[Dan_P]

Hello,

interesting, looking forward to your results!

What needs to be kept in mind is that Permanganate is not a very strong oxidizer - so not all organics will react. Very reactive is everything thats unsaturated.

What might be worth considering is switching from basic to acidic reaction conditions: Thus the reaction product should be likely Mn2+ ions instead of insoluble brown MnO4 - in addition the reaction mixture would likely be homogenous (the hydroxide precipitates magnesium salts), which might add some benefit in downstream photometry. But maybe there are also drawbacks, especially in seawater.

Edit: After thinking about it a bit longer it might not be a good idea, because Chlorine might be formed. If you want to try it, do it only at the microscale!

BR, Christoph

Agreed on small scale experimentation. No point in generating a bunch of chlorine under pressure.

 

[Dan_P]

Okay, this time the colors remained for all 4 samples.

I did not try a pure RO/DI sample before, so A is "new" to me. I prepared B and C just like before (just doubled the sample size and reagents). The only other difference is that I didn't use a syringe filter this time before adding NaOH and KMnO₄. Originally I used a Nylon 0.22µm filter. Maybe it released something into the samples which later on reacted with the KMnO₄ once I added the reagents?

I'm not yet sure if the colors make sense though.

• A is pure RO/DI and should have kept its color pretty well if I understand correctly what is happening. Maybe I grabbed the wrong canister and used RO/DI that has been sitting openly for a day.​
• B is freshly mixed 35ppt saltwater and I understand that it kept its color quite well (I would have expected A to look similar).​
• C is tank water and probably full of organics, so it is fair that it lost some of its color.​
• D is 1mL tank water and 9mL RO/DI and should be between A and C, but instead is more like B and C.​

Readings (zeroed with 35ppt freshly mixed saltwater):

• A: 1.44
• B: 1.70
• C: 0.75
• D: 1.19

Absolutely no syringe filters were used.

The chemistry in fresh water is at a higher pH than in saltwater because Mg(OH)2 precipitate removes base in saltwater. The color difference may also be a result of permanganate being convert to manganate and…I forget what other manganese species. So, ignore colors and results from freshwater tests

B and C are the expected results, though the DOC in your aquarium is like the DOC in my very wet skimmate samples. Keep in mind with this method that there is a possibility, though I suspect rare, that a little bit of very labile DOC can give a similar COD (permanganate consumption) as a high concentration of a rather inert DOC. I consider this unlikely because bacteria would never allow much labile DOC to exist for very long. The way that I confirm the COD measurement is with a BOD5 test, but that is for another day.

I suspect your skimmate is going to be potent. My skimmate COD is 50% from the particles (that’s the scum or mud you see accumulating) and 50% from the water.

 

[EnterName]

Okay, this time the colors remained for all 4 samples.

I did not try a pure RO/DI sample before, so A is "new" to me. I prepared B and C just like before (just doubled the sample size and reagents). The only other difference is that I didn't use a syringe filter this time before adding NaOH and KMnO₄. Originally I used a Nylon 0.22µm filter. Maybe it released something into the samples which later on reacted with the KMnO₄ once I added the reagents?

I'm not yet sure if the colors make sense though.

• A is pure RO/DI and should have kept its color pretty well if I understand correctly what is happening. Maybe I grabbed the wrong canister and used RO/DI that has been sitting openly for a day.​
• B is freshly mixed 35ppt saltwater and I understand that it kept its color quite well (I would have expected A to look similar).​
• C is tank water and probably full of organics, so it is fair that it lost some of its color.​
• D is 1mL tank water and 9mL RO/DI and should be between A and C, but instead is more like B and C.​

Readings (zeroed with 35ppt freshly mixed saltwater):

• A: 1.44
• B: 1.70
• C: 0.75
• D: 1.19

Absolutely no syringe filters were used.

The chemistry in fresh water is at a higher pH than in saltwater because Mg(OH)2 precipitate removes base in saltwater. The color difference may also be a result of permanganate being convert to manganate and…I forget what other manganese species. So, ignore colors and results from freshwater tests

B and C are the expected results, though the DOC in your aquarium is like the DOC in my very wet skimmate samples. Keep in mind with this method that there is a possibility, though I suspect rare, that a little bit of very labile DOC can give a similar COD (permanganate consumption) as a high concentration of a rather inert DOC. I consider this unlikely because bacteria would never allow much labile DOC to exist for very long. The way that I confirm the COD measurement is with a BOD5 test, but that is for another day.

I suspect your skimmate is going to be potent. My skimmate COD is 50% from the particles (that’s the scum or mud you see accumulating) and 50% from the water.

Just in case this helps to understand the measurements:

I'm currently dosing nutrients (phosphate, ammonia, and sometimes nitrate directly) because they drop to 0 otherwise.

The tank is approx 5 years old, understocked, and I'm lazy with water changes. ORP is low (300mV), UV is running, corals are happy. I assume I could get rid of carbon by keeping nitrogen and phosphate levels above a minimum... Might try aiming for higher levels soon.

I'm currently changing tanks and have to manually dose stuff until everything is set up, so I sometimes forget adding nutrients. The tank is prone to diatom growth, and I can't find the silicate source (5 stage RO, 3 stage DI unit, 0.053μS/cm @ 15°C should really be enough imo).

 

[EnterName]

Coming back with more "insight" or more confusion, let's see:

Experiment 1:
I wanted to see if the Nylon filters I used were the cause of the disappearing color, but the colors remained leaving me dumbstruck what on earth happened in my first experiments...

A: Non-filtered tank water: 1.83 (Yesterday's result of the same water but 5%-10% different sample size: 0.75)
B: Nylon 0.22µm filtered: 1.99
C: PES 0.22µm filtered: 1.88
D: Non-filtered 35ppt nyos pure mix: 2.29 (Yesterday's result of the same water but 5%-10% different sample size: 1.70)

A difference in sample size of 5%-10% between yesterday and today is expected as my measuring cylinder and the syringe I use for the initial water sample disagree on what "10mL" means :D.

1mL and 10mL pipettes are already ordered and will hopefully improve repeatability of results in the future.

Again all tests were zeroed with 35ppt nyos pure mix and I prepared 10mL samples with 2mL 2% NaOH and 2mL 390ppm KMnO₄.

----

Experiment 2:
I wanted to see how stable the readings were with the methods I'm using, so I used 10mL 35ppt nyos pure mix, added 2mL KMnO₄ without any NaOH or heating step, and measured the results directly:

Readings: 2.84, 2.94, 3.09, 3.08, 3.14, 3.03
n = 6 (sample size is small for now, I know)
Mean (µ) = 3.02
Standard deviation (σ) ≈ 0.11
Standard error of the mean (SEM) ≈ 0.05

Edit: The used vial was brand new and kept clean between tests with RO/DI. I ensured that no bubbles inside the vial or smudges on the glass could have interfered with the measurements.

----

Experiment 3:
Similar to Experiment 2 but this time with tank water and the whole process including heating, etc. No sample filtering. Each vial was prepared with 10mL tank water, 2mL 2% NaOH solution, and 2mL 390ppm KMnO₄ solution.

Readings: 1.76, 1.97, 1.92, 1.95
n = 4
Mean (µ) = 1.9
Standard deviation (σ) ≈ 0.1
Standard error of the mean (SEM) ≈ 0.05

 

[Dan_P]

Coming back with more "insight" or more confusion, let's see:

Experiment 1:
I wanted to see if the Nylon filters I used were the cause of the disappearing color, but the colors remained leaving me dumbstruck what on earth happened in my first experiments...

A: Non-filtered tank water: 1.83 (Yesterday's result of the same water but 5%-10% different sample size: 0.75)
B: Nylon 0.22µm filtered: 1.99
C: PES 0.22µm filtered: 1.88
D: Non-filtered 35ppt nyos pure mix: 2.29 (Yesterday's result of the same water but 5%-10% different sample size: 1.70)

A difference in sample size of 5%-10% between yesterday and today is expected as my measuring cylinder and the syringe I use for the initial water sample disagree on what "10mL" means :D.

1mL and 10mL pipettes are already ordered and will hopefully improve repeatability of results in the future.

Again all tests were zeroed with 35ppt nyos pure mix and I prepared 10mL samples with 2mL 2% NaOH and 2mL 390ppm KMnO₄.

----

Experiment 2:
I wanted to see how stable the readings were with the methods I'm using, so I used 10mL 35ppt nyos pure mix, added 2mL KMnO₄ without any NaOH or heating step, and measured the results directly:

Readings: 2.84, 2.94, 3.09, 3.08, 3.14, 3.03
n = 6 (sample size is small for now, I know)
Mean (µ) = 3.02
Standard deviation (σ) ≈ 0.11
Standard error of the mean (SEM) ≈ 0.05

Edit: The used vial was brand new and kept clean between tests with RO/DI. I ensured that no bubbles inside the vial or smudges on the glass could have interfered with the measurements.

----

Experiment 3:
Similar to Experiment 2 but this time with tank water and the whole process including heating, etc. No sample filtering. Each vial was prepared with 10mL tank water, 2mL 2% NaOH solution, and 2mL 390ppm KMnO₄ solution.

Readings: 1.76, 1.97, 1.92, 1.95
n = 4
Mean (µ) = 1.9
Standard deviation (σ) ≈ 0.1
Standard error of the mean (SEM) ≈ 0.05

Looks like the method is starting to behave.

I also switched to mechanical pipettes for better reproducibility.

 

[EnterName]

Looks like the method is starting to behave.

I also switched to mechanical pipettes for better reproducibility.

Yes, but I would have loved to understand what I did wrong the other day...

As soon as I find the time, I will be trying out different KMnO₄ concentrations to get the calibration curve you have been talking about.

 

[Dan_P]

Yes, but I would have loved to understand what I did wrong the other day...

As soon as I find the time, I will be trying out different KMnO₄ concentrations to get the calibration curve you have been talking about.

Debugging the past might not be as exciting as discovery :-)