Determination of Nitrate in Fresh Water Concentration of Samples by an Ion Exchange Procedure A. D. WESTLAND' and R. R. LANGFORDl Opeongo Limnological Laboratory, Department o f Lands and Forests, Province of Ontario, Whitney, Ont., Can.
An analg tical procedure has been developed which is applicable to determination of nitrate in fresh waters in a concentration range one order of magnitude lower than previous methods have allowed. Samples are concentrated by an ion exchange process before the strychnidine color determination is applied. Direct application of the color reaction to natural samples may lead to results which are much too high.
too much. Of the compounds tested, sulfanilic acid gave the best results. The final absorbance was influenced by the quantity of this reagent; therefore, it was added accurately from a micropipet. A study of the efficiency of the nitrite removal showed that 0.002 gram of sulfanilic acid was sufficient to eliminate interference from nitrite xvhen present in an amount comparable to the amount of nitrate. EXPERIMENTAL
A
STUDY of the biological productivity of lake waters necessitated a knowledge of the organic, ammonia, nitrite, and nitrate nitrogen in inland waters. Existing analytical procedures sufficed for the determination of the first three, but no method sufficiently sensitive for the estimation of the low concentrations of nitrate in northern Ontario waters has been reported. The most recent methods for nitrate (3,4),using the polarograph, are no more sensitive than older colorimetric ones. Natural water samples cannot be concentrated for nitrate determination by evaporation because the resulting decomposition and hydrolysis of organic nitrogen compounds vitiate determination of the original nitrate content. Previous studies on the concentration of phosphate samples by an ion exchange process ( 1 ) suggested the possibility of similarly concentrating nitrate samples. Nydahl ( 2 ) determined a variety of constituents following an ion exchange procedure, but gave little attention to nitrate It was felt that adsorption of nitrate on a chloride-form resin a t room temperature should be attended by little or no disruption of organic nitrogen compounds. The nitrate, along v i t h other strong acid radicals, should be eluted by a relatively small volume of elutriant, the concentration being then high enough for colorimetric determination. This was found to be the case and tenfold concentrations were effected. Preliminary examination of colorimetric methods for nitrate determination led the authors to choose strychnidine as a reagent, because this procedure of Zwicker and Robinson ( 5 ) gave the best results for low concentrations of nitrate. The strychnidine procedure suffers from the disadvantage that a curve, which does not obey Beer's law, must be obtained from standard samples determined simultaneously n ith each set of unknowns. Various techniques for carrying out the color development were investigated in order to establish conditions which gave the most reproducible absorbance values. The color intensity is sensitive to the method of mixing the color reagent with the sample solution and also to the time of standing, so that a precise manipulation technique had to be maintained. The mixing method described below gave reproducible results. The time allowed for color development was selected with regard t o the slow color development in samples of low concentration and fading in samples of high concentration. The choice of the chloride concentration of the solution was based on the study of the ion exchange process described further on. Because nitrite ion reacts with strychnidine to give an intense coloration, the possibility of eliminating i t by diazotization was investigated. The addition of a n amine reduces the nitrate color as well as the nitrite concentration, so a search was made for a compound which would not reduce the sensitivity for nitrate by 1 Present address, Department of Chemistry. University of Toronto, Toronto, Can. 9 Present address, Department of Zoology, University of Toronto, Toronto, Can.
Apparatus. COLORIJIETRIC.4 Rubicon Co. Evelyn photoelectric colorimeter equipped with a 540-mp filter was used for the absorbance measurements. The strongly acid atrychnidine reagent n a s delivered from an automatic buret greased n-ith silicone stopcock compound. The vent to the atmosphere was connected to a drying tower containing anhydrous magnesium perchloratme. IONEXCHASGE. Glass columns 0.8 cm. in inside diameter contained analytical grade Amberlite IR-4B ion exchange resin to a depth of 8 cm. The column effluent was delivered through a siphon tube 0.5 mm. in inside diameter. It n-as found that large bore tubing allowed back-diff usion of the solutions, thus preventing quantitative recovery by the necessarily small volume of elutriant used. Reagents. COLOPIMETRIC. For the color reagent, 1.0 m.V strychnidine solution was prepared by dissolving 0.320 gram of the reagent, obtained from Bios Laboratories, Ken. Tork, N . Y., in 1 liter of concentrated, low nitrat,e sulfuric acid. For sulfanilic acid solution, 0.6 gram of the reagent' (Distillation Products Industries, T o . T238) was dissolved in 70 ml. of hot water, 20 ml. of hot concentrated hydrochloric acid n-as added, and the volume adjusted to 100 ml. Standard nitrate solution 11-a~prepared by adding 0.722 gram of analytical grade potassium nitrate and 1 ml. of chloroform to sufficient water to make 1 liter of solution containing 100 p.p,m. of nitrate nitrogen. Dilutions to the desired concentration were made immediately before use. IONEXCHAXGE. Analytical grade sodium chloride solutions of various concentrations 17-ere used for elutions. The water used Tvas distilled from a Barnstead still and passed through a column of Amberlite IRA-400 ion exchange resin in the hydroxyl form t o produce anion-free water. In early experiments, large volumes of double-distilled r a t e r used for washing and preparing solutions introduced considerable nitrate and nitrite. This condition might have been remedied by distilling the water from alkaline medium, but the authors chose to use the more convenient but eff ect,ive device of further treating once-distilled mater by passing through Amberlite IR.4-400. Procedure. The p H value of a fresh sample is adjusted to between 5 and i with 0.01N acetic acid or sodium bicarbonate. If t,he chloride concentrat,ionis below 0.001.11, it is brought to this value by the addition of sodium chloride solution. The sample is passed at a rate of 1.0 to 1.5 ml. per minute through an Amberlite IR-4B column. The column is cleared of residual sample water hy passing through i t 25 ml. of anion-free water. The nitrate is eluted with 45 to 50 ml. of 1% sodium chloride solution and made up to 50.0-ml. A 5-ml. aliquot (which contains from 0.10 to 1.0 p.p.m. of nitrate nitrogen) of this solution is placed in a comparison tube. A 0.40-ml. volume of sdfanilic acid solution is added with mixing. The solut,ion is allowed t o stand for a t least, 10 minutes. Meanwhile, a 5.4-ml. volume of strychnidine reagent solution is delivered t o another comparison tube. The reagent is poured quickly but carefully into the sample, the solution is poured back into the first tube, and then forth and back once again. The color is allowed to develop for 20 l minutes and the absorbance is measured, using the 6-mm. aperture and the 540-mp filter. The sample should be protected from bright light. If several samples are to be determined a t once, a schedule of about one sample per minute should be followed during the color development. T o pre are the column for the next analysis, it is washed free of chlori& with 0.5 liter of anion-free water. Preparation of Standard Curve. Dilutions of the standard nitrate solution, containing from 0.1 t o 1.0 p,p.m. of nitrate nitrogen, were made. The dilutions were adjusted to contain 1% of
*
1996
1997
V O L U M E 2 8 , NO. 1 2 , D E C E M B E R 1 9 5 6 sodium chloride and the volume mas made up to 5 ml. Color was developed in the same manner as for the samples. RESULTS
Five independent analyses of a large sample of stream water, from which the precision of the method can be ascertained, appear in Table I . ’
Table 111.
Effect of Elutriant Chloride Concentration on Eluate Color and Nitrate Recovery (9.8 y of nitrate nitrogen taken) Sodium Absorbance Chloride, of Nitrate Recovered 5% Eluate Y 7% 0 25 0 50 1 0 2 0 3 0
Table I. Precision of Replicate Nitrate Determinations in Stream Water by Ion Exchange (500-ml. taken for each sample) Kitrate Sample Nitrogen KO. Concn., P.P.M. 1 2 3 4
0,058 0,061 0 062 0 061 0.057
5
DISCUSSIOl
In exploratory experiments, samples prepared by adding knoir-n amounts of the diluted nitrate and nitrite standards to 1 liter of the anion-free water were passed a t a rate of 1.0 to 1.5 ml. per minute through the column. The column was eluted a t the same rate with 45 ml. of 3% sodium chloride solution and the eluate made up to 50 ml. x i t h 3% sodium chloride solution. A 5-ml. aliquot m RF taken for analysis. The re.ultq of some determinations on such samples appear in Tablr 11. These shorn a slight tendency to positive errors, as blank solutions did also. This was a t first assumed t o be due to incomplete removal of nitrate in the water purification process, but the error TI as increased by slower passing of the eluting solution and I>> high concentrations of salt.
Table 11.
Recover>-of Sitrate from Synthetic Samples
Nitrite
Taken, Y
2.5 2.5 10 0 10 0 ~
5.0
Nitrate,
y
____._~___
Taken 10.0 10.0 10 0 10 0 10 0 20.0
Recovered 10.4 9.4 10 4 10 4 10 7 19.8
70
Recovery 104 94 104 104 107 99
I t was subsequently found that t,he resin m-as slightly dissolved by the elutriant to give a solution capable of forming a red color with strychnidine. I t was further noticed that a slight, brown color was imparted to elutriant solutions by the resin. To examine this quantitatively, it rvas necessary to measure the color of eluates using a 150-nim. layer of solution in a Lumetron Model 450 colorimeter with a 420-mp filter. The int.ensities of the color of eluates of various sodium chloride concentrations are listed in Table 111. Sitrate samples were passed through the columns and eluted Lvith solutions of the same chloride concentratione. The recoveries are recorded in the same table. An elutriant containing 1% sodium chloride gives quantitative recovery a n d dissolves only about half as much resin as t,he solutions used in the earlier experiments. A major consideration in the ion exchange process is the p H of the sample. The exchange process will not opera& if the p H is above 7. The waters dealt with in this laboratory are
0 014 0 026 0 032 0 045 0 060
2 0 8 4 9 9
11 2 10 1
20 86 101 114 103
almost invariably slightly acid, so that no adjustment of p H was usually necessary. Unless the sample was only feebly acid, the resin was partly dissolved; this process induced flocculation of the humic material in the resin bed. It v a s found advisable to keep the p H within the range from 5 to 7 . The adjustment with base or acid was not tedious because the organic acids present had a slight buffering effect. Weak acid or base should be used for this, otherwise hydrolysis of nitrogen compounds may occur. The choice of a chloride-form resin v a s opportune. -4 hydroxyl-form resin evokes a n abrupt change in acidity as the sample is passed through. The hydroxyl and the sulfate forms of the resin adsorb the tannins almost quantitatively, a hereas the extent of adsorption of the chloride-form resin is small. Any adsorbed and subsequently eluted organic nitrogen which can be hydro11 zed to nitrate by the strychnidine solution results in positive errors. The interference caused in this way was found to be roughly proportional to the color of the huniic matter in the eluate solution 17-hen measured 1~1tha 420-mp filter and a 150-mm. light path. Approximately 7% of the colored organic matter in 1%-aterobtained from a very dark stream was adsor bed on the chloride-form resin and was quantitatively eluted The effluent showed an apparent nitrate nitrogen concentration of 0 048 p p m. Assuming that the 7% of adsorbed organic matter could hydrolyze upon acidification t o a proportionate amount of nitrate, the error resulting mould be 0.048 p p.m. X 7/93 = 0.004 p.p.m. This is near the lower limit of the concentration range in hich the method lvould be applicable; therefore it mas desirable to decreaqe this error. It was discovered that if the sample were made to contain chloride in a concentration of O . O O l M , the extent of adsorption of organic matter was reduced to about two thirds of the former value. Higher concentrations effected no further decrease, however. The adsorption of nitrate from dark-colored natural stream nater was not inhibited by the presence of chloride in concentrations a t least as high as 0.002.11. The optical absorbance of the organic matter itself was negligible a t 540 mp. When the strychnidine procedure was applied to streain \vater fromwhich strong acid anions had been removed by passing through an Amberlite IR-4B column, a red color appeared indicating the presence of an oxidizing agent, probably nitrate. Presumably, this was not present as free nitrate ion during the exchange process but was released when the strong acid reagent was added. An attempt IT-as made to further justify this assumption. Dark-colored stream water was passed through the Amberlite IR-4B exchanger to free it from available nitrate. Known quantities of standard solution were added to the effluent water and the nitrate was then determined by the ion exchange concentrating procedure. Quantitative recoveries of the nitrate as reported in Table IV indicate the validity of the method. It is significant that when the strychnidine procedure was applied directly to the water samples-Le., omitting the ion exchange process-results high by several hundred per cent were often obtained. The same phenomenon was observed with phosphate when determined both directly with acid molyb-
ANALYTICAL CHEMISTRY
1998 Table IV.
Recovery of Kitrate Added to Blank Stream Water
tion sufficiently by coniplexing the chloride with mercury(I1) acetate. ACKNOF LEDGllENT
Color of
Sample so.
Feed Water, Absorbance Unit
Xitrate, y Added Recovered
0.010 0,010 0.010 0,057 0.057 0.057 0,057
2 11
5 05 5.05 5 05 5 05 10 6 10 6
2.16 5.85 4.84 4.93 5.08 11.3 10.5
%
Recovery 102 116
98 98
101 1Oi
99
The research n-as carried out with the support of the Research Division of the Department of Lands and Forests of the Province of Ontario. The authors wish particularly to express their gratitude to F. E. Beamish and IT.4. E. McBryde of the Department of Chemistry, University of Toronto, for their advice and encouragement. LITERATURE CITED
date and after an ion exchange concentration procedure similar to that for nitrate. The authors believe that the extent of the hydrolysis of organic nitrogen and phosphorus in conventional methods of analysis has not been sufficiently appreciated. No attempt was made to modify this procedure for application to sea water. If use of the ion exchange process for saline samples n-ere desired it might be possible to reduce the halide concentra-
(1) IIcBryde, IT.A. E., Harvey, K.. Uiiirersity of Toronto, private communication. (2) Sydahl, F., Proc. Intern. Assoc. Theor. A p p l . Limnol. 1 1 , 276 (1951). ~ . 25, 878 (1953). (3) Rand, hl. C., Heukelekian, H., - 4 ~ 4 CHEX (4) Salas, S. M. de, Rei,. obras sanit. nacidn (Ruenos Aires) 15, 23 (1951). (5) Zwicker, B. XI. G., Robinson, R. J., J . Marine Research (Sears Foundation) 5 , 214 (1944). RECEIVED for review January 16, 1956. Accepted June 13. 1956.
Calculation of Transfers Needed in Countercurrent Distribution ElNO NELSON School of Pharmacy, University of California M e d i c a l Center, San Francisco, Calif.
-4 method of calculating the number of transfers needed to effect a given degree of separation between a pair of solutes distributed in the countercurrent process is described. It is based on finding the number of transfers to obtain a given fraction of either solute between the peak of its distribution and the intersection of the distribution curves for the solutes distributed. -4pplications of the equations developed are illustrated and possible sources of error due to the method are discussed.
each solute is assumed to be distributed independently of the other and describable by Equation 1 n ith known values of X and 1'. Bfter the distribution of a pair-for example, solutes 1 and 2 with X and Y values, respectively, of X1, Yl and XZ, Y2-there will be some tube, r1, in n hich equal fractions of solutes 1 and 2 will be found. This corresponds to the intersection of the two distribution curves ( 6 ) . Therefore,
I T ~ , T =, ) (7'n>rt)* ~ from which on substitution with Equation 1 and solving for r % ,
S
EVERBL methods are available for calculating the number of
transfers needed to obtain a given degree of separation in the Craig countercurrent distribution process or for estimating the degree of separation obtained after a given number of transi ~ r sare applied. Among the methods available for calculating tJither or both of these quantities are those of Gregory and Craig ( d ) , Hecker ( S ) , Nichols (6),and Keisiger (6). I n determining the number of transfers needed to obtain a given degree of separation betveen a pair of solutes distributed, the number c ilculated will vary, depending on the method of calculation rhosen. This note describes a generally applicable method that is as accurate as possible when using Laplace's approximation to the binomial expansion that describes a countercurrent distribution. I n the Craig countercurrent distribution process, the distrihution of a solute may be described by
Tn,r = (n n!T ) ! T ! ( X ) T ( Y )
-
0)
where Tn,r is the fraction in tube T after n transfers, X is the fraction transferred Kith the moving phase (assumed to be the upper phase), and Y is the fraction remaining in the stationary phase (1). If a pair of solutes are distributed in the process,
The fact that Equation 1 is not defined for other than integral values of T results in no loss of generality. The fractions of each solute between the peaks of their distributions, n X 1and nX2, and r, ma)- be found by use of the follon-ing pquation and tabulated values for its integral ( 4 ) :
Tn,r
=
I \ ' 2 Tils Y
€
( n S - r)2 2nX y
-__-
(3)
This expression is generally accepted as a good approximation for Equation 1 when n is large and the quantity ( n X - r ) is small in relation to n X and nY. Here, ( n X - r , ) is the absolute number of tubes between the peak of the distribution and the tube corresponding to the intersection of the two distribution curves. Tabulated values of the integral of Equation 3 are so arranged that the tables are entered n-ith a number, (4)
and the area (fraction) betrreen nX and
T
is available directly.
