# Making Standards



## rjordan393 (Nov 23, 2012)

I would like to make 10 & 20 ppm NO3 standards and 1 & 2 ppm PO4 standards. After I make them and test them; can the test vials be sealed with something other then the caps that come with the test vials? I want to be able to use the tested standards as a reference color to my future tests and I do not want the standard vials to leak.
How long can I expect the tested standard to last?


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## AKnickolai (Nov 30, 2007)

PO4 standards are hard to make because you need to add so little mass wise to a liter of water to get in the 1-2ppm range. Your best bet is to use the Fertilator tool on this website. Enter what ever your sample size is going to be in the tank size section, then play around with the masses of KNO3 and whatever you use for PO4 until you reach the desired concentrations. Making one liter or gallon of reference solutions is probably as low a water volume as you'll want to go due to the small amounts of ferts required to make the solutions. I don't know how long the tested reference samples will keep. I usually make new reference solutions each time I want to do an accurate test.


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## Zapins (Jul 28, 2004)

Have a look at this thread I explained how to make standards here:
http://www.aquaticplantcentral.com/forumapc/fertilizing/89483-how-make-phosphate-solution.html

Also, I am unsure if you can save a vial of test liquid to compare with new tests. The reagents may keep reacting and change color.

Try it out though and let us know if the color stays the same, it would be quite useful if we could all make standards like that.


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## rjordan393 (Nov 23, 2012)

One way to find out is to call or email the manufacturer of the test kit. I use the "Nutrafin products by "Hagen". I'll email them. They are slow in replying based on my experience. So I do not expect an answer for about 3 weeks. I'll also do a search on my question.


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## Tugg (Jul 28, 2013)

I would also store in a cool dark place. Some reagents may be complex molecules and ambient light could potentially break them down with time.


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## rjordan393 (Nov 23, 2012)

Thanks, I am thinking the same thing; so I'll wait for "Hagen's" answer. I emailed them because I think I have a better chance for a response from a tech person instead of customer service by phone.
I just performed two tests and I am leaving the test vials out exposed to room light to see what happens over the course of 24 hours.


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## chibikaie (Nov 22, 2013)

If you have access to parafilm, it forms a better seal than the vial caps, which tend to be somewhat leaky. You would otherwise except some water loss over time to evaporation.

In the past, I have been extremely lazy about rinsing out test tubes, and have not noted a change in the final color even after several days. The same goes for used test strips (in my experience, test strips are as accurate or better than the liquid tests I have purchased, so I use them for routine testing).


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## rjordan393 (Nov 23, 2012)

Update:
My initial tests did not produce the desired results. I used Nutrafins NO3 & PO4 test kits.
The starting results indicated 7.5 ppm NO3 & 0.75 PO4. I left both test vials exposed to room light and temperature for 24 hours. I then checked for a color change. The results were an increase in color for both tests. NO3 was indicated at 20 ppm & PO4 was indicated at 1 ppm. So Tugg's response was right on.
But I think my next round of tests might produce the desired results. I have 8 ml clear bottles with screw caps on order. I intend to fill them with the tested standards to the very top, seal them with the screw cap so that no air can get in. Then place them in small box and keep them under refrigeration for 24 hours. Then check the results.
I have two graduated lab flasks to measure out my solutions and a jewelers scale that gives me readings of three decimal points to accurately weigh the chemicals.


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## Zapins (Jul 28, 2004)

Interesting results. Strange the readings didn't accurately measure 10 and 1 ppm to start with.


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## rjordan393 (Nov 23, 2012)

I believe you mean 7.5 ppm NO3 and 0.75 ppm PO4 to start with. After 24 hours, the NO3 in the vial was at 20 ppm and the PO4 was a 1 ppm. This may be understandable due to the vials being exposed to room light. temperature and oxygen for 24 hours. I forgot to mention these initial tests were samples of tank water.
I just tested again using the necessary equipment with the exception of using an oral baby syringe to measure out smaller ppm after making a 1000 ppm NO3 solution. I will see about ordering something more accurate then that. In the meantime, I will wait 24 hours to see if there is a color change.
This time, the tested standards were placed in 8 ml bottles filled to the top and sealed with a screw cap and refrigerated. Then check again each week.


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## Zapins (Jul 28, 2004)

You made 7.5 ppm standards? Why not 10 ppm? There is no 7.5 ppm on the test kits I have seen.


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## Tugg (Jul 28, 2013)

rjordan393 said:


> I forgot to mention these initial tests were samples of tank water.


This would make them useless as a test. Bacteria and organics in the water will breakdown and increase the levels. It could also go anaerobic if sealed and lower nitrate.

You need distilled water and inorganic fert salts for the test standards.


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## rjordan393 (Nov 23, 2012)

I think confusion has set in and maybe I was not clear in what I am trying to accomplish and that was making standards. But before I attempted that, I just wanted to see if there was a color change from a normal test of my tank water from its initial reading and then after 24 hours exposed to room temperature, light and oxygen. 
These initial tests from a sample of tank water were: NO3 was 7.5 ppm and was interpolated from the difference that I could see in color between 5 and 10 ppm on my color chart. After 24 hours, there was a color change and the vial indicated 20 ppm NO3. PO4 did not change as much. The test read 0.75 and was also interpolated. But after 24 hours, it looked closer to 1 ppm on my color chart.
I also noticed from past testing that when NO3 reads over 20 ppm, the interpretation is harder to determine on the chart. Then there's a difference in color depending on which light is being used. The test kit says to read the results with the light behind you. Which light? It does not say. I find there's a difference between taking a reading in incandescent vs outdoor light. Low NO3 readings of 5 to 10 ppm are not hard to interpret. But going beyond that is.
I believe this makes it all clear.

Yesterday, I received my 8 ml vials and proceeded to make my first standards of 10 & 20 ppm NO3. They are now in a small box in my refrigerator. I will check them 24 hours later and weekly for a color change. I used distilled water and hopefully not get a color change. After diluting 1000 ppm NO3 down to 50 ppm, I used a oral syringe (accuracy ??) as I did not have anything better to measure out the small amounts of NO3 solution to distilled water. The color of the standards when tested appear to be inline with the color chart. So I"ll have to wait and see what happens to the color over time. If I can achieve success in preserving my tested standards for 6 months, then it is worth my time. I will also look into purchasing something better then the oral syringe.


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## OTPT (Sep 27, 2010)

I think if you have a camera with an external flash.
You can place your vials containing the standard solutions 
in front of a white sheet of paper with the exteral flash behind
it (backlit) and take a photograph to use it as a reference.

Then when you want to compare your test result with 
the reference. Take a photograph of it and compare them 
in the computer. Cumbersome but in case the standards 
you make can't be preserved...

PS: shoot with camera and flash in manual mode.


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## rjordan393 (Nov 23, 2012)

I will leave that suggestion to another experimenter. I prefer to have the real thing in my hands. A picture would be similar to a color chart. 
If my experiment does not show promise, I will look into purchasing (if available) a device that comes with a circular disk that has differant hues of the same color. After the test vial is ready for viewing, its placed in the device and the disk is rotated until the hue matches the test vial.
A device such as this was available back in the late 80's and early 90's from the "Hach" company. But I think it has been discontinued.


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## JeffyFunk (Apr 6, 2006)

The preservation of 'developed' samples is not worth the trouble. From a chemist point of view, i c so many problems that it's not worth doing. 

Here's my recommendation. Make up standard solutions of Phosphate & Nitrate. According to standard methods, there is no recommended preservative for phosphate solutions so i'd use ~0.1% H2SO4 to reduce the pH < 2.0. For nitrates, the recommended preservative is either acid (if you aren't trying to determine nitrites, which i don't recommend getting into) or Chloroform (CHCl3) @ 2 mL per L. These standards should be stored in the refrigerator. Now, whenever you do an analysis, you should analyze one of the standard solutions and a blank in order to verify that your test kit is working properly. If it is not, then you have a positive and negative check and can determine what the problem is (usually it's contamination in the blank or a bad reagent, which would affect the color development of the standard). 

Now here is why i do not think it is a good idea to try to make up developed samples and 'preserve' them. For phosphate analysis, the samples are probably analyzed by the ascorbic acid method. These samples will continue to develop in color over time and so your color references will change as well. The method states that samples should be analyzed between 5 and 30 minutes after adding the reagents. 

I admit that i'm much less familiar with nitrate analysis, but looking at the recommended guidelines for sample collection for nitrate analysis (and nitrite analysis as well), these samples are simply prone to bacterial degredation (which is why the recommended preservative is chloroform). I just don't see samples of these being stable. 

As for nitrate analysis over 20 ppm, that's a problem with the sample, not the test kit. You are simply trying to analyze a sample outside of the accepted analytical range of the test kit and would need to dilute your sample to get it within range. Try taking 1 drop of the over range sample and adding 4 drops of DI water for a 20% dilution and checking the color then.


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## rjordan393 (Nov 23, 2012)

Thanks Jeff,
Its good to hear from someone with a Lab background. I was just about to make up some PO4 standards. So it appears that's the reason the test kit manufacturers give us charts, instead of glass vials of tested standards.
So another idea of mine is off the table. I"ll look into more professional type of kits for NO3 and
and PO4 from "Hach" and "LaMotte". I know the initial cost is high, but I want to see what the replacement reagents cost. I already seen some of the Hach kits with the color disk that is rotated to match the sample color. But they do not make it clear, what or how many reagents are needed to perform the Nitrate test. If they cost more then I pay for my otc Nutrafin test kit, then I'll just forget about purchasing a professional kit.
My Nutrafin kit for NO3 has a range of 5 to 110 ppm NO3.
I think the aquarium industry should provide us with the same type of disks as the professional kits provide. Then, I would be willing to pay more for a hobby kit.


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## Zapins (Jul 28, 2004)

Why not just buy a digital PO4 spectrophotometer? It will give you digital read outs to the 100th place and only costs $40. The nitrate spectrophotometer is more expensive though ($250).

http://reefbuilders.com/2010/04/20/...s-still-worth-the-resolution-and-portability/


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## rjordan393 (Nov 23, 2012)

Thanks,
I took a look at the PO4 low range pocket color meter (0.00 to 2.50 ppm range) and I am impressed. They also have a high range model (0.0 to 30 ppm).
In 2010, Hanna support said that they were looking into developing a pocket meter for NO3, but I see no mention of its availability. So I sent an email asking for a progress report. At present, their website is under construction. 
My NO3 tested standards are still under refrigeration and after 24 hours, I did not notice any change in color. But time will tell.


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## rjordan393 (Nov 23, 2012)

I got a reply from "Hagen" Tech. support stating that they feel that my tested NO3 standard will change color over time. Well I"ll have to see for myself how much time is needed. Its been 8 days since I made up two standards of 10 and 20 ppm NO3. 
Today, I tested my tank and got a reading of 10 ppm NO3 and then compared its color to my 10 ppm standard and they were a match.
I then increased the tanks NO3 level to 20 ppm and waited 45 minutes. I then tested and compared its color to my 20 ppm standard and this too was a match.
So far-so good. I am hoping to get about 3 to 6 months before any noticeable color change occurs. 
I also find that comparing my sample to the standards or the color chart is much easier to determine when they are taken outdoors for a reading. The refrigerated standards will develop condensation on the glass vial and should be wiped off for an accurate reading.
I used distilled water to make up my standards.

On my initial test, I used tank water and left the test vial exposed to room temperature air. After 24 hours, there was a significant increase in color in the NO3 sample.
So keeping the standards well sealed from air and under refrigeration, keeps the color stable up to now. I will be comparing my NO3 samples to my standards, once a month and will let all know what happens.


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## Zapins (Jul 28, 2004)

Good results so far. Did you fill the standards up to the lip and cap them without air inside?


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## rjordan393 (Nov 23, 2012)

Zapins said:


> Good results so far. Did you fill the standards up to the lip and cap them without air inside?


Yes, that I made sure of. when filling my vials, I filled them up to where they were about to spill over and then capped them.
I also keep the standards in a small box while under refrigeration.


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## Virc003 (Aug 18, 2011)

Back in my early college chemistry days, one of my profs mentioned using red cabbage extract as a universal pH indicator, and I went nuts with it. I made 5 or 6 vials to display the colored pH ranges. Unfortunately, the color faded within a day or two; however, acids and bases are quite different from inert fertilizers, so your standards might hold up.

My point is: why not make the standards from food coloring? This was the question given to me by my roommate as I was butchering the cabbage in our shared apartment kitchen. To a non-science major, all I was doing was mixing colors in bottles, but a color in a bottle is all you need, right?


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## Yo-han (Oct 15, 2010)

I'm not familiar with Hagens NO3 test, but most NO3 tests (using different shades of pink) state to compare colors after a x amount of time (usually 3 minutes). This is because it keeps changing color and the color comparison card is made for the 3 minutes mark. So this probably won't work. PO4 on the other hand is usually done with molybdenum, which turns blue immediate. This will probably be more stable, but will react with light and the container should be made of glass, because it will react with plastic too (plastic test vials will slowly become blue).

In the end, I think it is best to prepare reference solutions with RO water and PO4 and NO3 and check this at the start and when kept well sealed, you can compare later when you doubt your test kit or buy a new one! 

(I should have done that last one with my PO4 test kit, but this is another story...)


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## rjordan393 (Nov 23, 2012)

Update:

The 10 and 20 ppm nitrate standards do not have any color change after 35 days under refrigeration and kept in darkness in a small box. I did a nitrate test on my tank and based on my color chart, it was 7 ppm. it was more then 5 ppm but less then 10 ppm. I also placed my 10 ppm standard next to my tank sample to confirm my result. The tank sample was slightly lighter in color then my 10 ppm standard. The standard was also placed next to the color chart to double my confirmation.


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## rjordan393 (Nov 23, 2012)

Update:

The 10 and 20 ppm NO3 standards appear to be holding up after 2 months under refrigeration and darkness. My recent NO3 test indicated that I had a bit over 20 ppm in my 75 gallon tank. When I compared my test vial color to my 20 ppm standard, the test vial was a bit darker. But the real test comes after 6 months when I make new standards for both 10 and 20 ppm and then compare them to the original standards.


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## Zapins (Jul 28, 2004)

Very good to know.

So the standards do keep relatively stable long term. The 6 month mark will be the real test. If it works out I don't see why we shouldn't all make our own standards and keep them like you tested for comparison.


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## rjordan393 (Nov 23, 2012)

Today, I made up 1 and 2 ppm tested standards for phosphate. I have to see for myself if these will hold their color under the same conditions as the nitrate standards.


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## Tugg (Jul 28, 2013)

I'm curious as to how they would hold up at room temps with possible light. I don't suppose you left a set out to compare and see if refrigeration is even required?


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## rjordan393 (Nov 23, 2012)

No, I did not make up extra's. But after 6 months, I will make up new 10 and 20 ppm colored standards and then compare them to the old standards. If they match, then I will still keep the old under refrigeration and darkness to see if they will maintain the color for another 6 months. Then I can leave the new colored standards out at room temperature and exposed to light to see if this creates a change. So at the end of "May", the 6 month period will be over and I will make the new nitrate standards.


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## Zapins (Jul 28, 2004)

I think this is a really valuable experiment you are doing rjordan. All too often people don't calibrate their test kits and don't get accurate readings which leads to plant deficiencies. 

By the way I just noticed that you are fairly close to where I'm staying in Philadelphia (30-40 mins away). Do you know of any clubs in the area or decent fish stores? I have to admit I haven't really explored the area too well in the last year.


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## rjordan393 (Nov 23, 2012)

When I had a reef tank, I always patronized ""The Hidden Reef" which then was located in Northeast Philadelphia. But the store had a fire and the owner lost all his stock. They re-located to a much larger store less then a half mile from my house. It is located at 4501 New Falls Rd, Levittown, Pa. 19056. Its the first store I go to; not because its close but because they carry most of what I need and I find their knowledge of fish and aquariums exceeds those of other stores in the area. If I have a question, I always ask for "Jason". He is the manager.
Go to: http://thehiddenreef.com for more information and hours of operation.
I do not have any information on fish clubs.

If you decide to make a trip to the Hidden Reef, then if you want, you can stop by and take a look at my aquarium. I 'll send a PM and leave my phone number and address.


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## Phil Edwards (Jan 22, 2004)

To answer the earlier question; yes, N and P standards need to be kept refrigerated. Bacteria, molds, and fungi spores will get into the bottle during use and will throw the concentrations off. I *always* refrigerated my research standards for any material that is used by living things.


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