Reefkeeping 101 -
Chemistry - Adding Some Science to your Tank (Part 2)

Color My World

Last month we talked about instrumental and volumetric (drop count) methods for determining water quality. Salinity, pH, total dissolved solids (TDS), alkalinity and calcium are some of the most important test run on any marine tank and can be done instrumentally or by drop count. Ammonia, on the other hand, is not determined by drop count and, although there are instruments that can determine it, they are normally out of the financial means of the new aquarist.

Thankfully, in the mid 1800’s a whole group of German scientists found fairly simple ways of determining the concentration of a wide variety of substances. Historical names like Adolph Baeyer, August Beer, and Julius Nessler (Neßler actually) all were interested in dyes and how they reacted to light. Clothing dyes were a big business in those days and much research was centered on how colors were produced. Soon it was discovered that certain organic compounds and bases would react in a fixed fashion with other chemicals, or reagents.

Subsequently, the most popular method of testing is the use of colorimetric test kits. In these tests, a reagent is added to a fresh sample of tank water, which creates a color whose intensity increases in proportion to the concentration of substance that the test kit detects. This color increase is compared to a color standard chart that tells you how much of the compound is present in your water.

The simplest form of a colorimetric test is a test strip. It is just a strip of paper or plastic which has the reagents incorporated into the strip. You dip it into the water sample and it changes color so that you can compare it to a color chart or to bands of color already on the strip.

A step up is the color cube kit. Here you have a vial where you place the sample. You add the reagents, give it a shake, and then compare the solution with a colored plastic gradient for measurement. A Comparator and Disc test may also be used. Here you would put the sample in a device called a comparator, which has a split lens, one looking into the sample and the other at a disk with transparent colored filters. Much like the View-Master toy we all had as kids. You look through it and turn the disk until the colors match and then read the correlating measurements provided on the disc. Often the comparator has two tubes in the viewing field. In one side you place the sample and reagent(s) and in the other just a plain sample. This provides a means of viewing the color disks through a sample that also may have some color in it, say an algae bloom. When buying a comparator you want one that has fairly large tubes as the more sample one looks through the easier it is to determine lower levels of compounds.

Finally, for all you millionaire Newbie’s, there are instruments called filter photometers and spectrometers, which do the color matching electronically. The filter photometer uses colored filters that are turned into the light beam while the spectrometer uses a prism or diffraction grating to slit the light into the spectrum. This makes the spectrometer more versatile. If you are severely colorblind this may be the only method you can use. They are more technically complex and require some work to get good results but are more accurate. Unfortunately they are also more costly than other options I have mentioned, which makes their use less prevalent within the hobby.

You may have seen one of these (right), the ever popular Spectronic 20, in your high school chemistry lab. Once again you want one that can handle larger sample cells as it increases the path length and allows lower detection limits. The drawback of one like the Spectronic 20 is that you must construct a graph of light transmission vs concentration and that takes some work. Also, as instruments like the Spectronic 20 measure transmission, it is not a linear function and needs to be plotted on logarithmic chart paper. Upscale instruments use absorbance readings and that is linear, some actually do the calculations in the meter itself. Nice to have and they can be purchased at auction houses like labx.com.

Miller Time

There is a science principle on how these things work know as Beer’s Law. Now these drunken chemists were sitting at a bar one day and one had a full glass of beer in front of him. It was a pilsner and he was admiring the pretty yellow color looking down at the white bar countertop through the full glass of beer. Then one of his friends put another full glass right on top of his already full one. As he looked down, blurry eyed, through the two glasses of beer it dawned on him that the yellow color was twice as intense as it was before and he proclaimed to the bar, "If you double the amount of beer you look through, the intensity of the yellow color is twice as much as it is with one beer." So there you have it, Beer's Law. Double the concentration and you double the apparent color.

If you want to have some fun just post that explanation on Doc Randy's Chemistry Forum. It does explain the path length variable nicely however as if you look down at just one glass length of beer it is fainter in a yellow appearance than it you look at two stacked one upon the other. Double the path length and you double the color intensity.

It's Got Ammonia In It!

The most important colorimetric tests that a Newbie will use are the nitrogen tests: ammonia, nitrite and nitrate. Let's tackle ammonia first since it is the most important test, next to salinity, that one runs on a new tank. The test is simple. In an alkaline solution ammonia releases iodine from potassium iodide and produces a yellow to orange color. This is the Nessler test for ammonia. Low concentrations are light yellow and, as they increase, the intensity of the yellow increases until it becomes an orange color. Usually sodium hydroxide is used to get the very high pH needed for the test. This creates a problem as that both calcium and magnesium present in the salt water precipitate and make the sample cloudy. To prevent this, a chemical, often Rochelle salt (potassium sodium tartrate) is added to the sample. This ties up the calcium and magnesium and keeps them in solution. Because there is more calcium and magnesium in a reef versus a fresh water tank, a fresh water kit will not work on a saltwater tank. The precipitation inhibitor is not strong enough. If you use a freshwater kit on a marine sample you end up with a yellow milk shake so be sure to use the proper kit.

Another important thing with this, and most colorimetric tests, is there will be a waiting period for the color to develop. With Nessler reagent this takes from 8-12 minutes. It is important you time the test according to the manufacture's instructions. The color match, be it chart, cube or color wheel, is set to provide results only for tests run in the recommended time frame. Waiting too long can be just as bad as the color may continue to increase or in some cases fade.

Once the color is developed the sample is checked against the provided standards and the concentration read. Do the match against a well lit, white background if using cubes or wheels. Do not do it under something like a high K value MH or actinic lighting. That throws color rendition out the window. In fact, using light from a window is one of the best ways to view a colorimetric test. With the exception of the waiting period the ammonia test is fast and easy.

Some ammonia tests use the salicylate method. It works pretty much the same but the color produced has a greenish tint. In a solution with zero ammonia Nessler reagent is clear and salicylate is yellow. This method also produces somewhat higher results than the Nessler method. I prefer the Nessler method myself as it tends to only detect free ammonia, which is the most toxic in your tank. The salicylate method detects some bound ammonia which is not usually toxic. It is important if you compare test kits that use the same method or you end up comparing apples to oranges. Again, this applies to most kits. Different methods may produce different results because of the type of compound they detect.

Sometimes people test their RO/ DI water that has been stored for some time; lo and behold they find low levels of ammonia in it. This is not uncommon as ammonia is super soluble in water. Cleaning solutions, hair dyes and decomposition of materials all have ammonia. Since it is so soluble it will find its way into water with time. Unless you are stripping floor wax, which uses high concentrations of ammonia, the levels that you see are nothing to worry about.

This brings me to a point. Please be careful when handling test kit reagents. Some of these materials can be toxic. Don't leave them around where your little ones or pets can get a hold of them. For instance, the chemical in the ammonia test that creates the high pH needed is often sodium hydroxide. It is a fairly strong solution and can cause chemical burns and may be life threatening if swallowed. The kits made for the hobby are usually fairly safe but don't take any chances. DO NOT leave them lying about and be careful when handling them. Read the warnings that come with the kits and know the first aid measures needed if an accident occurs.

Ultimately you need to know how your ammonia test expresses results. The most common units are parts per million (ppm) and milliequivalents per liter (meq/l). It seems ppm is the unit most often used. Ammonia can be measured as pure ammonia (NH) or ammonia as nitrogen (N2). One ppm of ammonia N2 equals 1.3 ppm of ammonia NH3. Why have the ammonia as nitrogen N3 measured at all? It makes it simple to do calculation when ammonia and nitrate are both stated as nitrogen. If you do a mass balance then 12 ppm of ammonia as nitrogen will produce 12 ppm of nitrate as nitrogen (Note- this is rarely the case as some ammonia is converted to protein by organisms in the tank). Be sure to clarify which measurement your kit is using when asking for chemistry help on RC by saying ammonia as N or ammonia as NH3, that way the people who help you know right away what they are working with.

Here are some key points to remember when using color matching test kits:

• Get a representative sample. Don't use the little vial that comes with the kit. Get a measuring cup and get a sample below the water line in an area that has high flow like the sump return or near a powerhead. This water will be discarded after use.
• Rinse the sample cell several times with the tank water. DO NOT pour the rinse water back into the tank, as some reagents in the test may be toxic.
• Make sure your reagents are fresh. If you use liquid reagents you may wish to store them in the fridge. Make sure they are clearly marked. DO NOT store them in the fridge if you have youngsters in the house, PLEASE.
• Add the reagents to the sample in the order given and observe all time intervals, if any, between reagent additions. When using liquid reagents measure the amount added as the directions so indicate.
• Mix well. This is especially true when using powdered reagents. Make sure the powder is fully dissolved.
• Time the test. Allow the reagents to react according to the test kit instructions. Too short or long a period may give poor result.
• Use a white background in natural or incandescent light when matching colors. Never match colors with light from your tank lights or similar sources.
• Know the units you are using. This information is always provided in the kit, usually on the color chart.
• Do not let completed tests stand in the sample vial. Many reagents will stain the plastic or glass sometimes permanently. Pour out the sample promptly and rinse well with RO/DI water.

Night, Night Sweet Prince

Let's talk about nitrite-nitrate tests. I group them because they are basically the same test. Griess reagent, which is the color forming material in the test, only reacts with nitrites. To obtain nitrate results it must be converted to nitrite. Traditionally, the method was to expose the sample to copper plated cadmium granules. Since cadmium is so toxic, many kits now use an enzyme called Nitrate Reductase. In either case you want to shake your booty hard and for the time given in the directions. The nitrates in the water sample must come into contact with the granules to be reduced to nitrite. The reaction is immaterial, if nitrite or nitrate is present a red color results. If we run the test without the granules then we only obtain nitrite results. If we shake with the granules we get both nitrite and nitrate combined. Usually there are two separate test kits for these so you don’t have to worry about conversions and math. After the original tank set-up, few aquarists bother to test for nitrite. If you suspect you have nitrite but only have a nitrate kit you can qualitatively test for it by adding the reagents and immediately, without shaking, and get a red color. Finally, never rinse the sample cells in your tank water, as the reagents are toxic. Well, the sulfanilamide may help if you have a kidney infection. Only kidding folks, don't consume the test kit reagents.

A Flare for Phosphorus

Another popular test is for phosphates. Just like the nitrate tests above, the procedure is pretty much the same. The reagent(s) are added and a blue color is formed if phosphate is present. Interestingly the test reagents also react with silica and tests for that element use similar reagents. In the phosphate test an inhibitor is used to prevent the silica from reacting and in the silica test oxalic acid is added to break up the phosphate molybdenum complex that would interfere.

The worst thing about the phosphate test is that it is one of the easiest to screw up. Not because the test is hard but because stray phosphates are found all over the place, including detergents and tap water. Phosphates are real "sticky" and attach to glass like glue. To remove them requires some work. If you think your glassware is contaminated with phosphate get some muriatic acid (hydrochloric) from any pools supply store. Make a 50/50 mix (Caution corrosive) with RO/DI and let the glassware soak for a few hours. Then rinse well with RO/DI water. You want to mix this outside the house, as the fumes can be very irritating. Use phosphate test glassware exclusively for that test as many other tests kits use phosphates to buffer the pH to the proper level. If you forget and leave a completed phosphate test in the sample vial you may end up with blue glassware. Use the acid wash procedure above to remove it.

The common phosphate kits available usually test for only simple, orthophosphates. Complex phosphates, like hexametaphosphates, added to drinking water supplies, only show up if the sample is first boiled with acid. Organic phosphates require boiling with acid under oxidizing conditions. If you send a tank water sample to a private lab for total phosphate analysis don't be surprised if they report back levels while you were getting zero with your kit.

The Final Test

There is a whole range of metals that you can test by colorimetric methods. Only some are offered for the hobbyist's use but you can get others from the scientific supply houses. Copper is often tested when treating for ich and the test is pretty straight forward. There are so many other metal tests that I won't attempt to discuss them here.

Some people test for chlorine or chloramines. The method used here is known as the DPD method (you don't even WANT to know what DPD stands for). In it chlorine produces a pinkish red color and simple comparison is used. There are not too many (maybe none) kits offered for saltwater. I would guess the theory is that if you have measurable chlorine in your tank it has pretty much bought the farm. A FW total chlorine kit is handy if you worry that chlorine or chloramines are present in your RO/DI water. A total chlorine kit will detect chlorine, chloramines and chlorine dioxide; all common disinfectants in tap water. Since all are objectionable in your RO/DI water, which one it is matters little.

One of the major problems that many people face using colorimetric tests is that the test goes off the chart. All colorimetric test charts only show measurements within specific range. Take the phosphate test for example, as the phosphate concentration goes up the sample becomes deeper and deeper blue. Somewhere between 1.5 and 2 mg/l the blue becomes so intense that a large increase in phosphate creates a small increase in intensity of the blue color. In other words the test is no longer giving proportionate results. This level, the molar extinction limit, is where the test kit no longer works. There is a solution when this happens. Diluting the sample is all that is needed. You don’t need fancy lab equipment to do it. Just take a accurate measuring cup and add 1-cup sample to 1 cup RO/DI and repeat the test. Multiply the answer by 2 and you have the correct result.

Well, it is time to start thinking about Christmas shopping and ending this lecture. Have a happy holiday season and see you next year.

If you have any questions or comments about this article, please visit this thread in the New To The Hobby forum on Reef Central.