Water Research, Pergamon Press 1967. Vol. 1, pp. 205-216. Printed in Great Britain.

AUTOMATED ANALYSIS FOR NITRATE BY HYDRAZINE

REDUCTION

L. J. KAMPHAKE,S. A. HANNAHand J. M. COHEN

Engineering Activities, Research and Development, Federal Water Pollution Control Administration,
Robert A. Taft Sanitary Engineering Center, Cincinnati, Ohio, U.S.A.

(Received 16 September 1966)

Abstract--An automated procedure for the simultaneous determinations of nitrate and

nitrite in water is presented. Nitrite initially present in the sample is determined by a con-
ventional diazotization--coupling reaction. Nitrate in another portion of sample is quanti-
tatively reduced with hydrazine sulfate to nitrite which is then determined by the same diazo-
tization-coupling reaction. Subtracting the nitrite initially present in the sample from that
after reduction yields nitrite equivalent to nitrate initially in the sample. The rate of analysis
is 20 samples/hr. Applicable range of the described method is 0.05-10 rag/1 nitrite or nitrate
nitrogen; however, increased sensitivity can be obtained by suitable modifications.

I. I N T R O D U C T I O N

THE COMPOUNDSof nitrogen continue to be of interest to sanitary chemists because

of their importance in the life processes. Analyses for all or some of the forms of
nitrogen; ammonia, organic, nitrite and nitrate nitrogen, are commonly made to
monitor natural purification in streams, to control biological waste treatment, and to
determine compliance with nitrate standards for drinking water. All forms of nitrogen
are directly involved in the nitrogen transformations of decomposition-assimilation
and oxidation-reduction.
To properly understand these processes, sensitive and accurate analytical methods
are necessary for all forms of nitrogen involved. Methods which meet these criteria
are available for ammonia, organic and nitrite nitrogen forms. The analysis for
nitrate nitrogen, particularly in mixtures of nitrate-nitrite nitrogen, has, heretofore,
been difficult, principally because published procedures for nitrate nitrogen have
lacked either sensitivity or accuracy. They have also been subject to limitations from
interfering ions. This paper describes an automated procedure in which both nitrite
and nitrate nitrogen, alone and in mixtures, are determined simultaneously. The
method is accurate, precise and relatively free of interference from ions commonly
found in waters.
STANDARDMETHODS(1965) specifies two colorimetric methods for nitrate nitrogen,
but both have serious limitations. The method using phenoldisulfonie acid as the
color reagent is subject to interference from chloride and nitrite ions; the suggested
pretreatment for their removal frequently leads to inaccurate values. The brucine
method is more accurate, however, the developed color does not obey Beer's Law
and has the disadvantage that maximum absorbance occurs at a wavelength where
natural organic color in water can interfere. Other procedures based on polarography
and ultraviolet spectrophotometry have been inadequate because they are either non-
specific for nitrate ion or are subject to the same limitations described for the colori-
metric methods.
2O5
206 L.J. KAMPHAKE,S. A. HANNAHand J. M. COHEN
A procedure that utilized the sensitive and accurate colorimetric method already
available for nitrite ion appeared to have promise as a method for nitrate ion. The
successful approach lies in obtaining stoichiometric yields of nitrite ion in the reduction
of nitrate while maintaining conditions that do not, at the same time, reduce nitrite
ion. This approach has been described and used by others with varying degrees of success.
EDWARDS et al. (1962) described a manual procedure that used granular zinc to
reduce nitrate ion to nitrite ion after removing the initially present nitrite with azide.
The resulting nitrite ion was then determined colorimetrically. O'BRIEN and F~OR~
(1962) automated a procedure by pumping the sample through a column of granular
zinc. These two procedures do not provide stoichiometric yields of nitrite from
nitrate; unpredictable and varying reduction of nitrite decreases the precision and
accuracy of the analysis. The use of a column of zinc through which a large number
of samples are pumped has the disadvantage that the reduction performance of the
column varies with time and with the number and kind of samples. Reduction of both
nitrite and nitrate is affected.
Injection of a liquid reductant into each sample would intuitively appear to be
preferable to sample contact with a solid reductant whose activity could change with
time and sample composition. A promising liquid reductant was found to be hydra-
zinc. MULLIN and RILEY (1955) showed that hydrazine in alkaline solution produced
nitrite in good yield from nitrate. To avoid the concurrent reduction of nitrite,
FtsmaAy et al. (1964) first removed nitrite present initially in the sample and then
used hydrazine to convert the remaining nitrate to nitrite, but only 50 per cent yields
were obtained. For determining nitrate in sea water, HEtqRIKSEN(1965) used hydrazine
at 70C in an automated procedure. The yields of nitrite ranged from 85 per cent in
fresh water to 60 per cent in sea water. Moreover, he found that about 5 per cent of
the nitrite originally present in the samples was lost during reduction. The varying
yields of nitrite obtained in the reduction of nitrate and the concurrent loss of origin-
ally present nitrite required time-consuming corrections to obtain valid data.
Although these published procedures clearly demonstrated the applicability of hydra-
zine as a reductant, the optimum conditions for reduction had not yet been obtained.
An investigation of several variables in the hydrazine reduction step showed that
conditions can be controlled to provide stoichiometric yields of nitrite from nitrate
ion. Under these conditions, nitrite ion, initially present in the sample, is not reduced.
Hence, in an analysis for nitrite and nitrate ions having both present in a sample,
nitrite is determined separately on one portion of the sample by omitting the hydra-
zinc and using a colorimetric method. Hydrazine is added to another portion of the
sample, and the nitrite originally present combined with that formed from the reduc-
tion step is determined colorimetrically. The nitrate concentration is then obtained
by correcting for the nitrite concentration originally present in the sample.

2. AUTOMATED METHOD
Apparatus
A Technicon AutoAnalyzer* was used for the automation of the procedure. The
instrument uses a continuous flow system which automatically withdraws samples in
* Technicon AutoAnalyzer is manufactured by Technicon Controls, Inc., Chauncey, New York.
Mention of this product is for identification only and does not constitute endorsement by the Federal
Water Pollution Control Administration and the U.S. Department of the Interior.
 Automated Analysis for Nitrate by Hydrazine Reduction 207
succession from an array of sample containers; adds the appropriate reagents; and
passes the sample stream through a series of components for mixing, filtering and
heating. Absorbance of the color developed in the sample is displayed on a strip chart
recorder as a peak. Peak height which is proportional to concentration may be
evaluated by comparison with a calibration curve derived from standards.
The two manifolds required for the simultaneous determination of nitrite and
nitrate are shown in FIG. 1. After the sample is aspirated from the sampler module,
the sample is treated with sodium hydroxide and filtered to remove the ions which
would otherwise precipitate at the high pH needed for the reduction step. The filtrate
is split into two streams on manifold B, one remaining on manifold B for combined
nitrite and nitrate analysis ; the other stream is diluted with distilled water and pumped
back to manifold A for nitrite analysis. Sodium hydroxide is added to the nitrite
stream on manifold A followed by the acid color reagent. In the color reaction,
nitrite ion reacts with sulfanilamide under acid conditions to yield a diazo compound
which immediately reacts with N-l-naphthylethylenediamine forming a soluble red-
violet dye (JACOBS and HOCKHEISER, 1958). Absorbance, continuously measured in a
flow-through absorption cell at 540 m/~, is proportional to the concentration of nitrite
originally present in the sample.
Copper and hydrazine solutions are added to the other filtrate stream on manifold
B to reduce nitrate to nitrite. The flow is conducted through a heating bath in which
the temperature is adjusted to obtain optimum conditions for the reduction. The
stream now containing only nitrite ion reacts with the color reagent as above. The
resulting absorbance represents nitrite ion originally present and that formed from
the nitrate ion. The NO3-N content of the sample is obtained by subtracting the
original NO2-N from the total NO2-N.
Manifold-B Manifold-A
SAMPLER
I-I/2 mln

p.

120 AIR FILTER LMC OI Ol 290 SAMPLE

DI OI 2 90 DIS]~ H20 I 20 AIR
,~W.WASTE 0 80 0 3 2 I 0 N NoOH
0 42
- - - -
ic, 29 H2o
DO
01 DO DI DI 3 40 E u SOLON 160 COLOR REAG.
060 I0 N NoOH
I6 HYRAZlNE SOLON. f

Ao 12 A, R ~ ..... ~' 1

Ao I 60 COLOR REAG. -
2 50
LMC [
WASTE F -'-

Fro. 1. Diagram of manifolds.

Procedure
FIGURE 1 shows the assembled equipment. Baselines are established on both
recorders at 0.01 optical density while sampling distilled water. At a sampling time
of 1.5 min/sample, 20 analyses/hr can be made for both nitrite and nitrate. Each
208 L . J . KAMPHAKE, S. A . HANNAH a n d J. M . COHEN

standard tube and each sample tube is followed by a tube containing distilled water.
Because the success of the procedure requires that the peak heights resulting from
equal concentrations of NO2=N and NOa=N on the reduction side be equivalent,
temperature adjustments to obtain this equivalence must be made at the outset in the
following way. Two standards, one containing 2.0 mg/I NO2-N and another 2.0 mg/l
NOa-N, are passed through the system. The resulting two peaks must have equal
optical densities. Incomplete reduction is indicated when the NOa-N peak is lower
than the NO2-N peak. Equivalence is obtained by increasing the temperature of the
reduction bath. Conversely, if the NOa=N peak is higher than the N O 2 - N , IOSS Of
nitrite is indicated and the bath temperature should be decreased. The two standards
are reintroduced to determine whether equivalence has been achieved. Once the
correct temperature has been determined for the system and reagents, there is in-
frequent need for further adjustment.
The nitrite recorder side, manifold A using no reduction, should be observed for
the appearance of a single peak representing NO2-N only. The peak height for
NOz-N on this recorder need not be equivalent to the NO2-N on the recorder from
manifold B since different sample aliquots and reagent volumes are used.
The sampler drum is then loaded with a series of nitrite and nitrate standards to
obtain a calibration curve. The standards are followed by the samples making sure
that a tube of distilled water is inserted after every standard and sample to obtain the
necessary washout. Samples that have been preserved with acid should be neutralized
with dilute sodium hydroxide solution to approximately pH 7 before being placed in
the sampler. Single nitrite and nitrate standards are inserted after 10 samples as a
check on the proper operation of all components.
2 F No,-,.~o~-, RECORDER [2rI N O a - N RECORDER ~8
ZZ
[ MANIFOLD A
09,--
08--

o~- ~Z
Z
oo-

0.5-- --t

I
ZZ
I
I

02 ZZ I
I

o,~ ~ C

0.5 1.0 15 2.0 3.0 5.0 7.0 I0.0 0.5 t.0 1.5 2.0 3.0 5 0 7.0 tO.O
NITROGEN, m g / I NITROGEN,mg/I
FIo. 2. Optical densities of nitrate and nitrite standards from each recorder.
 Automated Analysis for Nitrate by Hydrazine Reduction 209
Examples of the calibration curves obtained on the two recorders are shown in
FIG. 2. It is important to note that peak heights for each concentration of NO2-N and
NOa-N from manifold B are equal and that only a single peak recording NO2-N is
observed from manifold A. The peak heights are not necessarily equal to the equiva-
lent concentration shown for manifold B.

Reagents
All reagents, except the color reagent, are prepared as concentrated stock solutions.
Working solutions are prepared by appropriately diluting the stock solutions. The
specified volumes permit continuous operation for at least 20 hr.
Color reagent. To approximately 3 1. of distilled water, add while stirring 400 ml
of concentrated phosphoric acid, 160 g of sulfanilamide and 8.0 g of N-l-naphthyl-
ethylenediamine dihydrochloride. Stir until dissolved, and dilute to 4 1. Store in dark.
This solution is stable for several months.
Stock hydrazine sulfate solution. Dissolve 54.0 g hydrazine sulfate, N2H4.H2SO4,
in 1800 mt of distilled water and dilute to 2 1. This stock is stable for 6 months.
Caution! Solution is toxic.
Working hydrazine sulfate solution. Using distilled water dilute 90 ml of stock
solution to 4 1. This dilute working solution is stable for several months without
special precautions.
Stock copper solution. Dissolve 2.5 g anhydrous cupric sulfate, CuSO4, in 1 1. of
distilled water.
Working copper solution. Dilute 25 ml stock copper solution to 4 1.
Stock sodium hydroxide solution (10 N). Dissolve 800 g NaOH in 1500 ml distilled
water. Cool and dilute to 2 1.
Working sodium hydroxide solution (1 N). Dilute 100 ml of l0 N to 1 1.
Stock nitrate-nitrogen standard (100 mg/1 NO3-N ). Dissolve 0.7218 g of potassium
nitrate, KNO3, and dilute to 1000 ml with distilled water. Preserve with 2 ml of
purified chloroform/1. The solution is stable for about 6 months.
Working nitrate-nitrogen standard. Prepare a series of working nitrate-nitrogen
standards, in the range 0 to 10 rag/l, by diluting stock. Preserve as above. Solution is
stable for about 6 months.
Stock nitrite-nitrogen standard (100 mg/1 NO2-N). Dissolve 0.4926 g sodium
nitrite, NaNO2, and dilute to 1000 ml with distilled water. Preserve as above. Solution
is stable for about 1 month.
Working nitrite-nitrogen standards. Prepare a series of working nitrite-nitrogen
standards of the same concentration as the nitrate-nitrogen standards. Preserve as
above. Solution is stable for about 1 month.
Purified chloroform. Interfering substances present in reagent grade chloroform can
be removed by extracting 1 1. of chloroform with five successive 200 ml portions of
0.15 N HC1.

3. EXPERIMENTAL RESULTS
Five factors that influence the reduction of nitrite and nitrate were investigated.
They were pH, hydrazine concentration, copper concentration and reduction time and
temperature. Some of these factors, as indicated below, were investigated by using a
gradient dilution technique as described by RYLANO et al. (1964). In this technique, a
210 L.J. KAMPHAKE,S. A. HANNAHand J. M. COHEN
constant flow of some reagent, for example sodium hydroxide, was added continuously
to a volume of distilled water which was kept well mixed. Concurrently, an equal flow
was constantly withdrawn from the distilled water--NaOH mixture, resulting in a
flow which contained a constantly increasing concentration of sodium hydroxide.

Effect of pH
Effect of pH on the reduction step was determined by continuously sampling a 10
mg/1 NO2-N or NOa-N standard while continuously increasing the pH with NaOH
using the gradient dilution technique described above. In these tests other factors
such as temperature, hydrazine and copper concentration and reduction time were
held constant. Since the color reaction must be conducted at strongly acid conditions,
pH 1.7 was maintained by injecting an increasing concentration of phosphoric acid
after the reduction bath, using the gradient dilution technique.
Reduction of nitrate to nitrite occurred above pH 11 and reached a maximum
optical density at pH 12.0-12.2. Color developed from the nitrite standard also
showed a slight increase above pH 11. However, the optical densities of equal con-
centrations of NO2-N and NOa-N were maximum and were similar only at pH
12.0-12.2.

Hydrazine concentration
The hydrazine sulfate concentration which would quantitatively reduce nitrate ion
but would not reduce nitrite ion was determined. In this test, two standard solutions,
10 rag/1 NO2-N and 10 mg/1 NOa-N, were sampled continuously, while a hydrazine
sulfate solution of increasing concentration was continuously being added. Other
[ 0 ,r
. . . . . . :,- . . . . .

-Iom~/~" / "'.'X

0,~ - -

0,5

0.4

0.3

cO

0,2

k.,
o.o J
tI _1 1~_
95 190 2~5 380 475
HYDRAZINE SULFATE, rng/I

FIG. 3. Effect of hydrazLqe sulfate concentration on reduction of nitrate and nitrite.

 Automated Analysis for Nitrate by Hydrazine Reduction 211
variables were held constant: Temperature 38C, reduction time (fixed by length of
heating coils and flow rate, approximately 10 min) and reduction p H 12.2. The
results are shown in FIG. 3.
The curve for nitrite ion shows a constant optical density of 0.92 as the hydrazine
sulfate concentration increases to 190 mg/1, at which point the optical density de-
creases sharply indicating reduction of nitrite ion. Reduction of nitrate ion starts with
the first small increment of hydrazine sulfate and steadily increases until quantitative
reduction is obtained at 190 mg/l. At this concentration, the optical densities of 10
rag~1 NO2-N and 10 mg/1 NOa-N are identical. Increasing the concentration of
hydrazine sulfate yields lesser optical densities on the NO3-N curve, since nitrite ion
formed from reduction of nitrate is simultaneously being further reduced. The stated
hydrazine concentration is a computed value of the concentration present in the
reduction volume.
A standard containing a mixture of 5 mg/1 NO2-N plus 5 rag/1 NO3-N treated with
hydrazine sulfate produced a maximum optical density of 0.92 with 190 mg/1 hydra-
zine, identical to the maximum shown in Fro. 3. The optimum hydrazine sulfate
concentration was independent of the concentration of either nitrite or nitrate within
the working range of the manifold. The optimum did change, however, as the tem-
perature and time of reduction were varied.
05 " ' - - q ' I

5mg/I
NO3 -N 1
o.31/
5 mg/I
NO2-N I
02[-
pH= 12:2
HYDRAZINE SULFATE =190 rng/I
REDUCTION TIME =lOmin
\

1
J
l

0.1
20
r
25
]
50 35
T

TEMPERATURE,C
40
! I
45 50
J
FIG. 4. The effect of temperature on reduction of nitrate and nitrite.

Temperature
Temperature is an important factor in the reduction of both nitrate and nitrite by
hydrazine. The effect of temperature is shown in Fro. 4. In this test all other factors
were held at their optimum ; p H 12.2, hydrazine sulfate concentration, 190 mg/1, and
212 L.J. KAMPHAKE,S. A. HANNAHand J. M. COHEN
time were constant; only the temperature of the reduction bath was varied between
20C and 52C. The temperature of the colorimetric step was held near ambient
temperature. The NOa-N curve shows that temperature in the range of 20-38C has
little effect (the slight rise in optical density is thought to be due to a rise in tem-
perature of the colorimetric step) but with increasing temperature, nitrite is rapidly
reduced becoming almost completely reduced at 49C. Although nitrate reduction
occurs at a temperature as low as 20C the reaction does not become quantitative
until within the range of 35-38C. Thereafter, the NOa-N curve slopes sharply down-
ward because of the simultaneous reduction of nitrite ion.
The sensitivity to temperature of the reduction of nitrate can be used to advantage
as a method to obtain coincidence of peak heights of nitrite and nitrate, thus com-
pensating for small changes in manifold conditions.

Copper catalyst
MULLIN and RILEY (1955) reported that a small amount of copper was necessary
as a catalyst to obtain reduction of nitrate ion with hydrazine sulfate but that a large
excess would interfere by precipitating as cuprous oxide.
The effect of copper ion concentration on the reduction of nitrite and nitrate is
shown in FIG. 5. Other conditions were held constant. Nitrite ion is completely un-
affected by copper ion concentration up to 1.0 mg/1 in the reduction mixture. The
NOa-N curve deafly shows that copper ion is necessary for the reduction reaction,
and that stoichiometric reduction is obtained at 0.5 mg/1. The reduction is not affected
by concentrations up to 1.0 rag/1. The latter concentration was selected for the
procedure.
1,0

0.3 v..,..lO nlg/t

Z NOa-N

O 0.2

pH=[2.2
0.1 TEMPERATURE -- 3 8 C
HYDRAZINE SULFATE=I90 m,::J/I
REDUCTION TIME = I0 rain

0.0
0.0 0,[ 0.2 03 0,4 0.5 0.6 0.7 0.8 09 1.0
Cu IN REDUCTION R E A C T I O N , m g / I

FIG. 5. Effect of concentration of copper ion on reduction of nitrate and nitrite.

 Automated Analysis for Nitrate by Hydrazine Reduction 213
Time of reduction
During development of the procedure, time of reduction was an important variable.
In all of the work reported here, time was held constant by using two coils in the
heating bath and by holding stream flows constant. Under these conditions, the time
of reduction was approximately 10 min. Acetone was used with manual procedures
to stop the reduction reaction but in this study this step was found to be unnecessary.

4. METHOD EVALUATION
Precision and accuracy
Precision was determined by replicate analyses of 25 1.00 mg/1 NO3-N and 25
10.0 mg/l N O a - N standards in distilled water, first in consecutive order and then in a
random distribution pattern. The results are shown in TABLE 1. Standard deviations

TABLE 1. SUMMARY OF STATISTICAL DATA

Analysis of samples in
Item
Consecutive order Random order
NOa-N taken (mg/1) 1.00 10.0 1.00 10.0
No. of determinations 25 25 25 25
Mean (mg/1) 1.00 10.0 1.00 10.0
Max. and min. 0.96-1.03 9.94-10.05 0.96-1.04 9.92-10.09
Standard deviation 0.018 0.027 0.024 0.073
95 ~ Confidence limit for one
determination 1.00 + 0.04 10.00+ 0.05 1.00+ 0.05 10.00+ 0.14

of 0.018 mg/1 and 0.027 mg/1 were obtained for 1.00 and 10.0 mg/1 NO3-N respec-
tively, when 25 consecutive determinations were made of each concentration. When
the concentrations were randomly distributed, the standard deviations were 0.024 and
0.073 mg/1 respectively for the same standards.
Accuracy of the method was evaluated by determining recovery of NOE-N and
NO3-N added in differing ratios to river water and raw domestic sewage. These data,
shown in TABLE2, indicate NO2-N recoveries ranging from 100.0-101.0 per cent and
NO3-N recoveries ranging from 100.3-106.0 per cent.

TABLE2. RECOVERY OF NITRATE AND NITRITE FROM R A W SEWAGE AND RIVER WATER

mg/l as nitrogen
Item
Raw sewage River water
Nitrite present* 0.00 0.00 0.02 0.02
Nitrate present* 0.09 0.09 0,28 0.28
Nitrite added 1.00 7.00 1,00 3.00
Nitrate added 7.00 1.00 3.00 1.00
Nitrite found* 1.00 7.05 1.02 3.05
Nitrate found* 7.11 1.12 3.43 1.34
Nitrite recovery 100.0 100.7 100.0 101.0
Nitrate recovery 100.3 103.0 105.0 106.0
* Average of 10 replicate analyses.
214 L.J. KAMPHAKE, S. A. HANNAHand J. M. COHEN
Interferences
Nine different ions commonly found in water and sewage were investigated for
possible interference in the colorimetric determination of nitrite and in the reduction
reaction of nitrate to nitrite. The gradient dilution technique served as a rapid means
for checking such interference. After a 10 mg/1 NO3-N or NO2-N standard had been
sampled continuously for 15 rain, a continuously increasing concentration of foreign
ion was introduced into the sample stream. The degree of interference could be
determined from any change in optical density for any calculated concentration of
added ion.

TABLE 3. IONS INVESTIGATED FOR POSSIBLE INTERFERENCE

rag/1 ion not producing interference

Ion Salt
Nitrate analysis Nitrite analysis
C1- NaCI 30,000 30,000
Mg2+ MgSO~ 75 75
Free chlorine -- 200 < 1.0"
NH3-N (NH,)2SO, 80 80
S2- Na2S "~ t
Ca 2+ CaSO, 240 240
Fe a + FeC13 30 30
1 D O 4 3 -- KH2PO,~ 50 50
ABS -- 60 60

* Chlorine oxidizes NO2 in sample to NOa.

-~Apparent NO2 and NO3 varied + 10 per cent up to 10 mg/1 of sulfide ion.

Interference in either the colorimetric or reduction steps was not observed at con-
centrations below those listed in TAnLE 3. Up to 30,000 mg/l of chloride ion did not
interfere. Chlorine oxidized nitrite in the standards to nitrate which was quantitatively
recovered upon reduction with hydrazine. Nitrate was not affected by chlorine in
concentrations up to 200 mg/1. Sufide ion in all concentrations up to I0 rag/1 caused
random variations of about _+ 10 per cent in both the nitrite and nitrate concentrations.
Concentrations of magnesium and calcium, higher than those shown, tended to plug
the filter paper with their precipitates.
Analytical grade chloroform, commonly used as a preservative, in a concentration
of 1 ml/100 ml of sample showed an interference which was equivalent to approxi-
mately 0.05 mg/l NOa-N. This interference can be eliminated by purifying the
chloroform as described previously under Reagents.

Sensitivity
The manifolds described here were designed for a range of 0.05 to 10.0 mg/1 NO3-N
or NO2-N. Under the optimum conditions for reduction and colorimetric reactions,
0.05 rag/1 yields an absorbance of 0.005. Greater sensitivity may be obtained by:
(a) replacing the 8 m m colorimetric absorption cell with either a 15-mm or a 50-ram
 Automated Analysis for Nitrate by Hydrazine Reduction 215
cell ; (b) using a range expander on the recorder; or (c) increasing the size of the pump
tubes that pick up the flow from the filter. Increasing the cell light path to 50 ram, for
example, will increase the sensitivity to a range of 0.01-3.0 mg/1. If the method to
increase the size of the pump tubes is selected, then the rate of pumping 1 N NaOH
to each manifold should be reduced proportionately to compensate for the additional
NaOH present in the larger sample aliquot.

Practical application
The impetus to develop a procedure for nitrate ion that was superior to those
available arose from the analytic requirements of a study on the transformations of
various nitrogen forms in an impounded water. In this study, SYMONSet al. (1965)
added nitrogen in the form of ammonium ion to a nutrient medium which was then
seeded with microorganisms. Analyses were made at intervals for ammonia, nitrite,
nitrate and organic nitrogen. A basic requirement of this research was that, in the
absence of nitrogen lost as gases from the system, the sum of these forms had to be
unvarying throughout the experiment.
During the early stages of the study, when nitrate nitrogen was being determined by
reduction to nitrite ion using a zinc column (O'BRIEN and FiORE, 1962) anomalies
were observed in the data. This difficulty was attributed to the nitrate analysis and
specifically to the zinc column reduction step.

24 I ~ 1 7 [ [ T
~N -
20
x. NHa-N / NO3-N
18-- \ /
"tt /
Z 16 -- .\ /
E J4 -- "\ i

g
':-
6 -~- NO2-N/I.
'/ X i \

J ,c 3 ORG N
ol.-..~,~" I _ I "~--~--A.-d....~._.A I 1
0 2 4 6 8 10 12 14
TIME, doys
FIG. 6. Nitrogen species formed during nitrification o f a m m o n i a (SVMONS et al., 1965).

Use of the present procedure in this study has largely eliminated the described
difficulty. A typical set of curves showing the transformations that nitrogen forms
undergo in the experimental medium is shown in FIG. 6. The curve of interest here is
the nitrogen summation curve which showed an average deviation of 1.6 per cent
from the mean in the 14-day experiment during which the NO3-N concentration varied
from 0-20 mg/l. The present procedure has been used successfully for the above type
of analyses and others for over 2 years.
C W
216 L . J . KAMPHAKE,S. A. HANNAHand J. M. COHEN
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FISHMAN M. J., SKOUGSTADM. W. and SeARBROG. F. (1964) Diazotization method for nitrate and
nitrite. J. Am. Wat. Wks Ass. 56, 633-638.
HENRIKSEN A. (1965) An automatic method for determining nitrate and nitrite in fresh and saline
waters. Analyst 90, 83-88.
JACOBS M. B. and Hocr~qEISER S. (1958) Continuous sampling and ultramicrodetermination of
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MULLIN J. B. and RILEY J. P. (1955) The spectrophotometric determination of nitrate in natural
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O'BRIEN J. E. and F I o ~ J. (1962) "Robot Chemist" determines nitrates in sewage and wastes.
Wastes Engng 33, 128-132.
RYLAND A. L., PICKHARDTW. P. and LEWISC. D. (1964) Automation techniques in analytical research.
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STANDARD METHODSFOR THE EXAMINATIONOF WATER AND WASTE WATER (1965) 12th ed. pp. 195-
204. American Public Health Association, New York.
SYMONS J. M., WEIBEL S. R. and ROBECK G. G. (1965) Impoundment influences on water quality.
J. Am. Wat. Wks Ass. 57, 51-75.