Analytica Chimica Acta, 114 (1980) 191-197

0 ElsevierScientific PublishingCompany, Amsterdam -Printed in The Netherlands

SIMULTANElOUS DETERMINATION OF NITRATE AND NITRITE BY

FLOW INJECTION ANALYSIS

M. F. GIN& H. BERGAMIN FO*, E. A. G. ZAGATTO and B. F. REIS

Centro de Energia Nuclear na Agricultum, CEP. 13.400 Piracicaba. S. Paul0 (Brasi!)
(Received 27th June 1979)

SUMMARY

An automatic method for the simultaneous determination of nitrate and nitrite by

flow injection analysisis described. Nitrate is reduced to nitritewith a copperized cadmium
column. Nitrite is diazotized and coupled with N-(1-naphtbyl)ethylenediammonium
dichloride. The merging zones approach is used to minimize reagent consumption. The
injector system is arrangedso that two peaks are obtained, one corresponding to nitrite
and tbe other to nitrite plus nitrate_ A sampling rate of about 90 samples per hour is
possible; the precision is better than 0.5% for nitrite in tbe range 0.1-0.5 mg 1-I and
1.5% for nitrate in tbe range 1.0-5.0 mgl-.

Since its inception in 1975 [I], flow injection analysis has undergone fast
development. Devices such as special injection valves [ 141, dampeners [ 51,
solvent extraction chambers [ 31, distillation units [6], etc., have been
designed with a view to automation of many different types of analysis. The
concept of merging zones in flow injection analysis was introduced in 1978
[ 71. The injector port had then the function of introducing simultaneously
both sample and reagent into the analytical system. This led to a drastic
reduction of the reagent consumed [7, 81, allowed the analysis of acidic
samples without pre-neutralization [9], and, in connection with a stopped-
flow procedure, was employed in enzymatic assays based on slow reactions
[lo]. The use of this technique to perform standard addition, and also to
improve sensitivity in turbidimetry, is a common practice in this laboratory.
Another function which can be performed by the injector port is the
commutation of parts of the manifold during sample and reagent injection.
Development of flow injection malysers based on this idea have opened up
new possibilities for carrying out simultaneous determinations without
splitting, blank corrections, assays of very variable samples, etc.
The main purpose of this paper is to describe a method for the simul-
taneous determination of nitrate and nitrite in soil and water samples, by
using a spectrophotometric method for nitrite and reduction of nitrate to
nitrite with a copperized cadmium column [ll, 121. A special injector-
commutator which operates in two positions is described. In one position,
the sample is injected and the column is placed in the analytical line, the
192

resulting signal corresponding to nitrate plus nitrite. In the other position,

the sample is injected again but the column is bypassed, so that the signal
corresponds to nitrite alone. The merging zones approach is used to mini-
mize reagent consumption.

EXPERIMENTAL

Flow system
The flow injection system without sample splitting is outlined in Fig. 1.
The sample and reagent are injected into corresponding carrier streams. At
point X, the sample zone meets the reagent Rl, which acts as reduction
catalyst, masking agent [IS] and buffer. After coil CI, the sample zone enters
the reduction column, nitrate being reduced to nitrite. At point Y, the sample
zone meets the colour-forming reagent, RI, starting the diazotization and
coupling reactions. After the reaction coil, Cz , the absorbance corresponding
to nitrite (original plus reduced nitrate) is measured at 540 nm. In the
alternative mode (dotted lines in the Bow diagram), the column is replaced
by simple tubing, and only the nitrite originally present in the sample is
measured.
The flow rates indicated in Fig. 1 were fixed to permit the analysis of
90 samples per hour with adequate sample dilution by the confluent streams.
The optimum pumping rate of the colour reagent RI was established by
using a 0.2 ppm N (as nitrite) solution in an infinite volume situation 163
and increasing the flow rate from 0.23 to 1.6 ml mm-l; maximum absor-
bance was found for the range 0.9-U ml min-.
The peristaltic pump, tubing, connectors, flow cell, spectrophotometer
and recorder were the same as used in earlier work [S 3 . The reduction
column was made from a glass tube (3 mm i-d.) filled with copper&d
cadmium filings, held in position by glass wool plugs.
The injector-commutator (Fig. 2) was made from perspex and had charac-

-_

Fig. 1. Flow diagram of the system_ CS (water) and C RI (8% phosphoric acid) are the
carrier streams corresponding to the sample (S) and reagent (R,). R, is the masking,
buffering and catalytic reagent, PP is the peristaltic pump; flow rates are given in ml
min-. C,, C, and C, are coils with lengths of 15, 150 and 15 cm, respectively. The
column is placed between coils C, and C,. D is the detection unit and W denotes waste.
For deta.iIs, see text.
 193

I _i
Fig. 2. Schematic diagram of the injector commutator in the position for nitrate plus
nitrite analysis. a and b are the sample loops, c is the reduction column, d and e are
COnneCtOrs, and f and g are the reagent loops_ M represents the manifold. The other
symbols are explained in the text.

teristics similar to those of the multiple proportional injector [S] . In the

position shown in Fig. 2, the sample (S) is aspirated to fill the sample loop
(b), the &our-forming reagent (RI)ispumped to fill the reagent loop (g),
and these loops define exactly the sample and reagent volumes. The excess
of sample goes to waste (W), while the excess of reagent, slightly diluted by
the reagent carrier stream, is directed to the reagent recovery vessel (V). In
this position, the reduction column (c) is placed in the analytical line. The
previously injected sample and reagent aliquots (a, f) are transported for
analysis by the corresponding carrier streams (C, and C, ,). In the other
position, the sample and reagent are aspirated again and the entire process
is similar, except thaq the reduction column is replaced by tube (d).

Reagents
All reagents were of analytical grade and distilled-deionized water was
used in all experiments. Copperized cadmium filings were prepared daily
by treating cadmirun filings (20-40 mesh) as described elsewhere [12].
The colour reagent R1 was prepared by dissolving 20 g of sulphanilamide
and 1 g of N-(1-naphthyl)-ethylenediammonium dichloride in 100 ml of
80% phosphoric acid and diluting to 1 1 with water. This solution was
stored in a retigerator. Reagent R2 was prepared by dissolving 100 g of
ammonium chloride, 20 g of sodium tetraborate and 1 g of NatEDTA in 1 1
of water; this solution is stable.

Samples and standards

Water samples from the Piracicaba region were collected in polyethylene
vessels and analyzed immediately. Soil extracts were obtained as described
by Bremner [13]_
The aqueous 1000 ppm N03-N stock solution (6.07 g NaN03 I-) and
1000 ppm NO&N stock solution (4.92 g NaNOz I-) were treated with a few
194

drops of chloroform and kept in a refrigerator. The nitrite solution was

standardized against a 0.1 N permanganate solution.
Working standards containing both nitrate and nitrite were prepared by
appropriate dilutions and covered the ranges 0.0-0.5 ppm NOir_N and O.O-
5.0 ppm NO?N. For soil analysis, the standards were diluted with the corres-
ponding extractant. Only one standard containing 0.50 ppm NO;-N is
necessary for calculation purposes; a 0.50 ppm NO;-N standard is useful for
checking the reduction conditions.

Optimization of the flow system

The infinite volume situation [6] was employed to define the optimum
reaction coil length. With the system indicated in Fig. 1, the column was
bypassed and a 0.2 ppm NOz-N solution replaced the sample carrier stream.
Coil lengths of 20, 44,94,148,200 and 250 cm were tested for coil CZ. For
each situation, the percentage reaction was determined by stopping the
pump after achievement of the plateau, allowing full development of the
reaction in the flow cell (Fig. 3). A 150cm coil permitted 80% of the com-
plete reaction. Longer coils provided better sensitivity but required larger
sample volumes [ 143, causing a significant decrease in the sampling rate.
The optimum length of the reduction column was established similarly
by using a 0.2 ppm NO:-N solution, the reaction coil being 150 cm. Reduction
column lengths of 2.0, 3.5 and 10.7 cm were tested (Fig. 4). The injector-
commutator allowed the columns to be replaced easily without disturbing
the system_ For each situation, the percentage reduction was determined by
comparison of the plateau achieved with that corresponding to a 0.2 ppm
NO?-N solution processed in the same way. A column length of 5.2 cm was

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Fig. 3. The influence of the reaction coil length. Curves a-f correspond to coil lengths
of 20,44,94,148,200 and 250 cm, respectively. The parallel lines on the right indicate
the steady-state. The break points correspond to pump stoppage, and the lines to the left
show the reaction development in a static situation. Paper speed, 0.5 cm min-.

Fig. 4. The influence of tbe reduction column length. From left t6 right,-the peaks (in
triplicate) correspond to column lengths of 10.7,3.5 and 2.0 cm, respectively.
 195

chosen, because the reduction was almost complete and reproducibility was
good.
With these dimensions of the system, the sample injection volumes were
fixed as 75 ~1 and 15~1 in order to give suitable measuring conditions. The
synchronization coil C3 (15 cm) and injected volume of reagent (50 ~1) were
adjusted to incorporate the merging zones approach in the proposed method.
The dispersion factors were estimated with a 0.2 ppm NOT-N standard, as
described earlier 1143.

RESULTS AND DISCUSSION

Some analytical characteristics of the proposed method are apparent from

the calibration data given in Fig. 5. As each position of the commutator
defines a different system, two baselines are observed. For pure nitrate
standards, no signal was recorded when the system was in the nitrite analysis
position, but of course when pure nitrite standards were used, signals were
obtained in both positions (Fig. 5d). This must be taken into account in
calculating nitrate concentrations. The net peak height for nitrate is calcu-
lated from the expression: H (nitrate) = H (total) - CYH (nitrite), where H
represents peak height; o! is defined as the ratio between the two peak
heights corresponding to nitrite alone with and without the reductor column
and must be determined before any routine run (Fig. 6). From the cali-
bration runs in Fig. 5, this ratio was calculated as 0.131, with a coefficient of
variation of 4.3%.
The difference in the peak heights of the same standard (Fig. 5d) reflects
the different dispersion in each analytical position; this is mainly due to the
different injected volumes and not to the presence of the column. The

0.5 -

10 20 cm

Fig. 5. Characteristics of the proposed system. (a) Nitrate standards (5.0, 4.0, 3.0, 2.0,
1.0, 0.0 ppm NOT-N). (b) Mixed standards containing nitrate (5.0,4.0, 3.0,2.0,1.0 ppm
NOTN) plus 0.5 ppm NO>N; the four small peaks to the right correspond to a standard
cont&&g 1 ppm NO;-N without nitrite and to a blank injection. (c) Mixed standards
containing nitrite (1.0, 0.8, 0.6, 0.4, 0.2, 0.0 ppm NOi-N) plus 2.5 ppm NOT-N. (d)
Nitrite standards (1.0-0.0 ppm NOTN). Paper speed, 0.5 cm miC_
196

0.5

00

Fig. 6. Routine run of seven water samples with standard addition_ From left to right:
a 0.5 ppm NOT-N standard followed by a 0.5 ppm NOT-N standard; a calibration run with
mixed standards (5.0-1.0 ppm NO;-N plus 0.5-0.1 ppm NO;-N); seven samples with
standard addition; seven original samples; and the calibration graph again. Paper speed,
0.5 cm min-.

column appears to have very little effect on sample dispersion. The measured
dispersion factors were 0.35 and 0.05, allowing an increase in sensitivity by
injecting larger sample volumes.
On a routine basis, 75 samples per hour were analyzed for both anions
(Fig. 6), with a relative standard deviation of 1.5% and 0.5% for nitrate and
nitrite, respectively. A sampling rate of about 90 samples per hour is possible
with a carryover less than 1%.
The results of the recovery tests made with water samples and soil extracts
are shown in Table 1.The sample showing low nitrite recovery was abnor-
mally coloured; the effect is not apparent in the nitrate recovery because of
the higher sample dispersion.

TABLE 1

Recovery data related to water and soil samples

Water Soil extra&C

[NOT-N] Recoveryb [NO;-N] Recoveryb [NO;-N] Recove&
(ppm) (S) (ppm) (X) (ppm) (W)

0.11 102.6 0.84 98.3 4.39 98.8

0.10 101.8 0.88 97.9 3.82 95.2
0.07 99.8 0.17 100.4 1.97 98.0
0.07 88.0 0.17 98.6 3.80 102.1
0.25 99.7 0.55 97.3 2.22 100.4
0.17 100.0 0.46 99.8 4.02 98.9
0.40 100.5 0.57 98.3 5.43 95.1

%Yhe NOT-N contents of the soil extracts were below 0.05 ppm. bAfter addition of 5 ml
of a 10.0 ppm NO;-N plus 1.0 ppm NO;-N solution to 45 ml of sample. =After addition
of 5 mi of a 30.0 ppm NO;-N to 45 ml of sample.
 197

The utilization of the two alternative systems allows the simultaneous

determination of two species present in different concentration ranges in the
sample with only one detector. There is no loss of reproducibility or
reduction in the sampling rate, such as occur when sample splitting is used.

The authors thank Drs. E. H. Hansen and J. RiiBiEka for providing the
reagents and Peter B. Vose for assistance in the preparation of the original
manuscript. Partial support of this project by FINEP (Financiadora de
Estudos e Projetos, Brasil) and by the Secretaria da IndGstria, CiGncia e
Tecnologia do Estado de S5o Paulo (Brasil) is greatly appreciated.

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