ALBUMINOID NITROGEN.
The addition of an alkaline permanganate solution to liquids containing nitrogenous organic matter causes the formation of ammonia, which can be distilled and determined by Nesslerization of the distillate. The nitrogen of the ammonia, thus obtained, is called albuminoid nitrogen. As the ratio of nitrogenous organic matter to the ammonia obtained by distillation is decidedly variable[[6]][[30]][[75]] in sewages and other substances containing much nitrogenous organic matter albuminoid nitrogen results on such substances are less accurate[[29]] than organic (Kjeldahl) nitrogen. Therefore in sewage work, including analysis of influents and effluents of purification plants and the water of highly polluted streams, it is recommended that determinations of organic nitrogen be substituted for determinations of albuminoid nitrogen. For ground waters and surface waters containing but little pollution, the albuminoid nitrogen is approximately one-half the organic nitrogen; accordingly the continuance of albuminoid nitrogen determinations for this class of work is approved.
Reagents.—Alkaline potassium permanganate. Pour 1,200 cc. of distilled water into a porcelain dish holding 2,500 cc., boil 10 minutes, and turn off the gas. Add 16 grams of C. P. potassium permanganate and stir until solution is complete. Then add 800 cc. of 50 per cent clarified solution of potassium hydroxide or an equivalent amount of sodium hydroxide and enough distilled water to fill the dish. Boil down to 2,000 cc. Test this solution for ammonia by making a blank determination. Correct determinations by the amount of this blank.
Procedure.—After the collection of the distillate for ammonia nitrogen described on page [15] add 50 cc. (or more if necessary to insure the complete oxidation of the organic matter) of alkaline potassium permanganate and continue the distillation until at least four portions, and preferably five portions, of 50 cc. each, of distillate have been collected in separate tubes. Determine the albuminoid nitrogen in the distillate by Nesslerization. If the albuminoid nitrogen is known to be high it is convenient to collect the distillate in a 200 cc. flask and to Nesslerize an aliquot part of it.
Dissolved albuminoid nitrogen may be determined in a sample from which suspended matter has been removed by filtration either through filter paper or through a Berkefeld filter. Suspended albuminoid nitrogen is the difference between the total and the dissolved albuminoid nitrogen.
ORGANIC NITROGEN.[[24b]][[69]][[71]][[76]][[84]]
Procedure for water.—Boil 500 cc. of the sample in a round-bottomed flask to remove ammonia nitrogen. This usually causes the loss of 200 cc. of the sample, which may be collected for the determination of ammonia nitrogen. Add 5 cc. of nitrogen-free concentrated sulfuric acid and a small piece of ignited pumice. Mix by shaking and place over a flame under a hood. Digest until copious fumes of sulfuric acid are given off and the liquid finally becomes colorless or pale straw color. Remove from the flame, and add potassium permanganate crystals in small portions until a heavy green precipitate persists in the liquid. Cool. Dilute to about 300 cc. with ammonia-free water. Make alkaline with 10 per cent ammonia-free sodium hydroxide. Distill the ammonia, collect the distillate in Nessler tubes, Nesslerize, and compare with standards as described (pp. [16]–18).
First procedure for sewage[[76]].—Distill the ammonia nitrogen directly from 100 cc. or less of the sample, diluted to 500 cc. with nitrogen-free water. Collect the distillate and determine the ammonia nitrogen in it. Add 5 cc. of nitrogen-free sulfuric acid and 1 cc. of 10 per cent nitrogen-free copper sulfate, and digest the liquid for half an hour after it has become colorless or pale straw color. Add 0.5 gram of potassium permanganate crystals to the hot acid solution, and dilute to 500 cc. with ammonia-free water. Dilute 10 cc. or more of this liquid, in a Kjeldahl distilling flask, to about 300 cc. with ammonia-free water. Make alkaline with 10 per cent sodium hydroxide, distill, and Nesslerize. With some samples direct Nesslerization may be used. (See p. [19].)
In this determination care must be taken to digest thoroughly, to add potassium permanganate to the point of precipitation, to sample carefully after dilution, and to add enough sodium hydroxide to insure the separation of the ammonia from the precipitated manganese hydroxide. Potassium permanganate should not be added during digestion because it causes loss of nitrogen.
Second procedure for sewage.—Omit the separation of ammonia nitrogen and determine the ammonia nitrogen and organic nitrogen together. Determine the ammonia nitrogen in a separate sample by direct Nesslerization as described on page [19]. The organic nitrogen is equal to the difference.
NITRITE NITROGEN.[[51]][[63a]][[64]][[94c]][[108]]
Reagents.—1. Sulfanilic acid solution. Dissolve 8.00 grams of the purest sulfanilic acid in 1,000 cc. of 5 N acetic acid (sp. gr. 1.041) or in 1,000 cc. of water containing 50 cc. of concentrated hydrochloric acid. This is practically a saturated solution.
2. α-naphthylamine acetate or chloride solution. Dissolve 5.00 grams solid α-naphthylamine in 1,000 cc. of 5 N acetic acid or in 1,000 cc. of water containing 8 cc. of concentrated hydrochloric acid. Filter the solution through washed absorbent cotton or an alundum filter.
3. Sodium nitrite stock solution. Dissolve 1.1 gram silver nitrite in nitrite-free water; precipitate the silver with sodium chloride solution and dilute the whole to 1 liter.
4. Standard sodium nitrite solution. Dilute 100 cc. of solution 3 to 1 liter, then dilute 50 cc. of this solution to 1 liter with sterilized nitrite-free water, add 1 cc. of chloroform, and preserve in a sterilized bottle. One cc. = 0.0005 mg. nitrogen.
5. Fuchsine solution. 0.1 gram per liter.
Procedure.—Place in a standard Nessler tube 50 cc. of the sample, decolorized if necessary with nitrite-free aluminium hydroxide (see p. [42]) or a smaller amount diluted to 50 cc. At the same time prepare in Nessler tubes a set of standards, by diluting to 50 cc. with nitrite-free water, various amounts of the standard nitrite solution. The following amounts of standard solution are suggested: 0.0, 0.1, 0.2, 0.4, 0.7, 1.0, 1.4, 1.7, 2.0, and 2.5 cc. Add 1 cc. of the sulfanilic acid solution and 1 cc. of the α-naphthylamine acetate or hydrochloride solution to the sample and to each standard. Mix thoroughly and allow to stand 10 minutes; then compare the sample with the standards. Do not allow the sample to stand more than one-half hour before making the comparison. If the color of the sample is deeper than that of the highest standard repeat the test on a diluted sample. If 50 cc. of the sample is used 0.01 times the number of cc. of the standard matched equals parts per million of nitrite nitrogen. Satisfactory results can be obtained by using either hydrochloric or acetic acid in preparing the test solutions, but the speed of the reaction is more rapid if acetic acid is used.[[112]]
Permanent standards may be prepared by matching the nitrite standards with dilutions of the fuchsine solution. Fuchsine standards have been found to be sufficiently accurate for waters high in nitrite and for sewage. The standards should be checked once a month and kept out of bright sunlight.
NITRATE NITROGEN.[[16]][[36]][[90]][[100]]
Two methods are recommended for the determination of nitrate nitrogen in water, sewage, and sewage effluents.
PHENOLDISULFONIC ACID METHOD.[[1]][[5]][[32]]
Reagents.—1. Phenoldisulfonic acid. Dissolve 25 grams of pure white phenol in 150 cc. of pure concentrated sulfuric acid. Add 75 cc. of fuming sulfuric acid (15 per cent SO3), stir well, and heat for 2 hours at about 100°C.
2. Potassium hydroxide solution. Prepare an approximately 12 N solution, 10 cc. of which will neutralize about 4 cc. of the phenoldisulfonic acid.
3. Standard nitrate solution. Dissolve 0.72 gram of pure recrystallized potassium nitrate in 1 liter of distilled water. Evaporate cautiously to dryness 10 cc. of the solution on the water bath. Moisten residue quickly and thoroughly with 2 cc. of phenoldisulfonic acid and dilute to 1 liter. This is the standard solution, 1 cc. of which equals 0.001 mg. of nitrate nitrogen.
4. Standard silver sulfate solution. Dissolve 4.4 grams of silver sulfate free from nitrate in 1 liter of water. One cc. of this solution is equal to 1 mg. of chloride.
Procedure.—The alkalinity, chloride, and nitrite content, and color of the sample must first be determined. If the sample is highly colored decolorize it with freshly precipitated aluminium hydroxide. Measure into an evaporating dish 100 cc. of the sample, or if nitrate is very high such volume as will contain about 0.01 mg. of nitrate nitrogen. Add sufficient N/50 sulfuric acid nearly to neutralize the alkalinity. Then add sufficient standard silver sulfate to precipitate all but about 0.1 mg. of chloride. The removal of chloride may be omitted if the sample contains less than 30 parts per million of chloride. Heat the mixture to boiling, add a little aluminium hydroxide, stir, filter, and wash with small amounts of hot water. Evaporate the filtrate to dryness, and add 2 cc. of the phenoldisulfonic acid, rubbing with a glass rod to insure intimate contact. If the residue becomes packed or appears vitreous because of the presence of much iron, heat the dish on the water bath for a few minutes. Dilute the mixture with distilled water, and add slowly a strong solution of potassium hydroxide or ammonium hydroxide until the maximum color is developed. Transfer the solution to a Nessler tube, filtering if necessary. If nitrate is present a yellow color will be formed. Compare the color with that of standards[[52]][[55]] made by adding 2 cc. of strong potassium hydroxide or ammonium hydroxide to various amounts of standard nitrate solution and diluting them to 50 cc. in Nessler tubes. The following amounts of standard nitrate solution are suggested: 0, 0.5, 1.0, 1.5, 2.0, 4.0, 6.0, 8.0, 10.0, 15.0, 20.0, and 40.0 cc. These standards may be kept several weeks without deterioration. If 100 cc. of water is used the number of cubic centimeters of the standard multiplied by 0.01 is equal to parts per million of nitrate nitrogen.
Standards that will remain permanent for several years if stored in the dark may be prepared from tripotassium nitrophenoldisulfonate.[[5]]
If nitrite nitrogen is present in excess of 1 part per million it should be oxidized by heating the samples a few minutes with a few drops of hydrogen peroxide free from nitrate repeatedly added[[95]] or by adding dilute potassium permanganate in the cold until a faint pink coloration appears; the nitrogen equivalent of the nitrite thus oxidized to nitrate is then subtracted from the final nitrate nitrogen reading.
REDUCTION METHOD.[[2]][[46]]
Reagents.—1. Sodium or potassium hydroxide solution. Dissolve 250 grams of the hydroxide in 1.25 liters of distilled water. Add several strips of aluminium foil and allow the evolution of hydrogen to continue over night. Concentrate the solution to 1 liter by boiling.
2. Aluminium foil. Use strips of pure aluminium about 10 cm. long, 6 mm. wide, and 0.33 mm. thick and weighing about 0.5 gram.
Procedure.—To 100 cc. of the sample in a 300 cc. casserole add 2 cc. of the hydroxide solution and concentrate by boiling to about 20 cc. Pour the contents of the casserole into a test tube about 16 cm. long and 3 cm. in diameter, or of approximately 100 cc. capacity. Rinse the casserole several times with nitrogen-free water and add the rinse water to the liquid already in the tube, thus making the contents of the tube approximately 75 cc. Add a strip of aluminium foil. Close the tube by means of a rubber stopper through which passes a bent glass tube about 5 mm. in diameter. Put the shorter arm of the tube flush with the lower side of the rubber stopper and let the longer arm extend below the surface of distilled water in another test tube. This apparatus serves as a trap through which the evolved hydrogen escapes freely. The small amount of ammonia escaping into the trap may be neglected. Allow the action to proceed for a minimum period of four hours or over night. Pour the contents of the tube into a distilling flask, dilute with 250 cc. of ammonia-free water, distill, collect the distillate in Nessler tubes, and Nesslerize. If the nitrate content is high collect the distillate in a 200 cc. flask and Nesslerize an aliquot part. If the supernatant liquid in the reduction tube is clear and colorless the solution may be diluted to a definite volume and an aliquot part Nesslerized without distillation.
TOTAL NITROGEN.[[93]]
In sewage work it is frequently of assistance to know the total nitrogen content. This is ordinarily computed by adding together the organic, ammonia, nitrite, and nitrate nitrogen, each of which is determined as already described.
OXYGEN CONSUMED.[[24]][[67]][[84a]][[85]][[94f]][[101]][[102]]
Oxygen consumed means the oxygen that the oxidizable compounds of sewage and water consume when treated in an acid solution with potassium permanganate. The expression is synonymous with oxygen required, oxygen absorbed, and oxygen-consuming capacity. It should not be confused with biochemical oxygen demand.
As the carbon, not the nitrogen, in organic matter is oxidized by potassium permanganate, oxygen consumed is considered by some an indication of the amount of carbonaceous organic matter present. The determination indicates, however, only part of the carbon, the proportion varying in different samples because the carbon in nitrogenous matter is not so readily oxidized as that in carbonaceous organic matter. Furthermore, it does not directly differentiate the carbon present in unstable organic matter from that in fairly stable organic matter, such as is sometimes referred to as residual humus matter. As nitrite nitrogen, ferrous iron, sulfide, and other oxidizable mineral substances reduce potassium permanganate, corrections for them should be made in the determination.
RECOMMENDED METHOD.
Reagents.—1. Dilute sulfuric acid. Dilute 1 part of concentrated sulfuric acid with 3 parts of distilled water and free the solution from oxidizable matter by adding potassium permanganate until a faint pink color persists after the solution has stood several hours.
2. Standard ammonium oxalate. Dissolve 0.888 gram of the pure salt in 1 liter of distilled water. One cc. is equivalent to 0.1 mg. of oxygen. An equivalent quantity of oxalic acid or sodium oxalate may be used.
3. Standard potassium permanganate. Dissolve 0.4 gram of the crystallized salt in 1 liter of distilled water. Add 10 cc. of the dilute sulfuric acid and 10 cc. of this solution of potassium permanganate to 100 cc. of distilled water, and digest 30 minutes. Add 10 cc. of the ammonium oxalate solution, and then add potassium permanganate till a pink coloration appears. This destroys the oxygen-consuming capacity of the water used. Now add another 10 cc. of ammonium oxalate solution and titrate with potassium permanganate. Adjust the potassium permanganate solution so that 1 cc. is equivalent to 1 cc. of ammonium oxalate solution or 0.1 mg. of available oxygen.
Acid digestion.—Place in a flask 100 cc. of the water, or, if the water is of high organic content, a smaller portion diluted to 100 cc. Add 10 cc. of sulfuric acid solution and 10 cc. of standard potassium permanganate and digest the liquid exactly 30 minutes in a bath of boiling water the level of which is kept above the level of the contents of the flask.[[70]][[71a]] If the quantity of permanganate is insufficient for complete oxidation repeat the digestion with a larger quantity; at least 5 cc. excess of the standard permanganate should be present when the ammonium oxalate solution is added. Remove the flask, add 10 cc. of the ammonium oxalate solution, and titrate with the standard permanganate until a faint but distinct color is obtained. If 100 cc. of water is used the number of cubic centimeters of potassium permanganate solution in excess of the number of cubic centimeters of ammonium oxalate solution is equal to parts per million of oxygen consumed.
If oxidizable mineral substances, such as ferrous iron, sulfide, or nitrite, are present in the sample corrections should be applied as accurately as possible by suitable procedures. Direct titration of the acidified sample in the cold, using a three-minute period of digestion, serves this purpose quite well for polluted surface waters and fairly well for purified sewage effluents. Few raw sewages containing no trade wastes need such a correction, but raw sewages containing “pickling” liquors do need it. If the sample contains both oxidizable mineral compounds and gaseous organic substances the latter should be driven off by heat and the sample allowed to cool before applying this test for the correction factor. If such corrections are made the fact should be stated with the amount of correction.
Period and temperature of digestion.—As the practice in regard to the period and temperature of digestion has varied widely it is difficult to compare the results obtained at one laboratory with those obtained at another. None of the methods gives absolute results. They are all relative[[26]][[29]][[57]] at best. Digesting 30 minutes at the boiling temperature is herein designated the recommended method. If samples are analyzed by any other method the method should be noted, and, representative results by the standard method should be placed on record for purposes of comparison.
OTHER METHODS.
Additional reagents.—1. Potassium iodide solution. Ten per cent solution, free from iodate.
2. Standard sodium thiosulfate. Dissolve 1.0 gram of the pure crystallized salt in 1 liter of distilled water. Standardize this solution against the standard potassium permanganate. As the thiosulfate solution does not keep well determine its actual strength at frequent intervals.
3. Starch indicator. Prepare as directed in the section on dissolved oxygen (pp. [65]–66).
4. Sodium hydroxide solution. Dissolve 1 part of pure sodium hydroxide in 2 parts of distilled water.
Certain widely practiced deviations from the standard procedure just described are noted in the following paragraphs.
1. Heat the acidified sample to boiling, add the permanganate solution, and digest for two minutes[[16]] at boiling temperature. This procedure is facilitated by agitating the liquid constantly with a small current of air to guard against bumping.
2. Same method as No. 1 except that the period of digestion is five minutes.[[121a]]
3. Same method as No. 2 except that the permanganate solution is added to the acidified sample when cold, and digestion is continued five minutes after the sample reaches the boiling point. The advantage of this method is that there is included the oxygen-consuming power of the volatile matter present in some sewages and sewage effluents, which is driven off by heat and thus escapes when the test is made in accordance with procedures 1 and 2.
4. Same method as No. 3 except that the period of digestion is 10 minutes.[[63]][[68c]]
5. Digestion of the sample after the acid and permanganate solutions are added is carried out abroad, especially in England, at approximately the room temperature,[[24a]][[69a]][[94f]][[100a]] apparently to guard against decomposition[[17]] of permanganate in the presence of high chloride, for periods of three minutes, fifteen minutes, and four hours; many observers record the oxygen consumed after all three periods, while some record the result only for the four-hour period. At the end of the period of digestion, add 0.5 cc. of potassium iodide solution to discharge the pink color; mix; titrate the liberated iodine with thiosulfate until the yellow color is nearly destroyed, then add a few drops of starch solution and continue titration until the blue color is just discharged. The number of cubic centimeters of potassium permanganate solution in excess of the number of cubic centimeters of sodium thiosulfate solution is equal to parts per million of oxygen consumed.
6. Digestion in alkaline solution[[104]] is preferable to digestion in acid solution for brines or waters high in chlorine. Place in a flask 100 cc. of the sample, or if it is of high organic content a smaller portion diluted to 100 cc. Add 0.5 cc. of sodium hydroxide solution and 10 cc. of standard potassium permanganate and digest exactly 30 minutes. Remove the flask, add 5 cc. of sulfuric acid and 10 cc. of the standard ammonium oxalate, and titrate with the standard potassium permanganate as in the acid digestion.