Verification of spectrophotometric method for nitrate analysis in water samples

Puji Kurniawati, Reny Gusrianti, Bledug Bernanti Dwisiwi, Tri Esti Purbaningtias, and Bayu Wiyantoko

Citation: AIP Conference Proceedings 1911, 020012 (2017);

View online: https://doi.org/10.1063/1.5016005
View Table of Contents: http://aip.scitation.org/toc/apc/1911/1
Published by the American Institute of Physics
 Verification of Spectrophotometric Method for Nitrate
Analysis in Water Samples

Puji Kurniawati1,a), Reny Gusrianti1), Bledug Bernanti Dwisiwi2), Tri Esti

Purbaningtias1), Bayu Wiyantoko1)

1
Professional Program of Chemical Analyst, Islamic University of Indonesia
Gedung FMIPA Kampus Terpadu UII Jl. Klaiurang Km 14,5 Sleman, Yogyakarta,
2
Environmental Agency of DI Yogyakarta Province
a)
Corresponding author: puji_kurniawati@uii.ac.id

Abstract. The aim of this research was to verify the spectrophotometric method to analyze nitrate in water samples using
APHA 2012 Section 4500 NO3-B method. The verification parameters used were: linearity, method detection limit, level
of quantitation, level of linearity, accuracy and precision. Linearity was obtained by using 0 to 50 mg/L nitrate standard
solution and the correlation coefficient of standard calibration linear regression equation was 0.9981. The method
detection limit (MDL) was defined as 0,1294 mg/L and limit of quantitation (LOQ) was 0,4117 mg/L. The result of a
level of linearity (LOL) was 50 mg/L and nitrate concentration 10 to 50 mg/L was linear with a level of confidence was
99%. The accuracy was determined through recovery value was 109.1907%. The precision value was observed using %
relative standard deviation (%RSD) from repeatability and its result was 1.0886%. The tested performance criteria
showed that the methodology was verified under the laboratory conditions.

INTRODUCTION
Nitrate (NO3-) is a natural inorganic ion and it included in the nitrogen cycle. Nitrate ions are often found in
ground water and surface water because nitrate is the result of oxidation of nitrite. Nitrate is tasteless, odorless, and
colorless in water, so it is undetectable without testing [1]. Nitrate-containing compounds in the soil are usually
soluble in water and can migrate with underground water. The nitrate content in the water spring depends on the
activity that produces pollutants on upstream, well water use activity, the industrial waste, the septic tank, the animal
waste, the use of fertilizers and pesticides on the plant around the well water. Some of these activities can cause the
nitrate content in good water is high.
The normal limit of nitrate content in clean water according to minister of Health Regulation of Indonesia
No.416/1990[2] is 50 mg/L and in drinking water is 10 mg/L. Nitrate in drinking water can be harmful to infants and
young children, lead to Methemoglobinemia and increased risk of spontaneous abortion or certain birth defect on
pregnant women if it exceeds 10 mg/liter [3]. The effect of high nitrate concentration can cause ovarian and bladder
cancer, cardiovascular disease, stomach cancer, miscarriage, high blood pressure, the formation of nitrosamine and
bad effect on the nervous system [1, 3, 7, 8].
Spectrophotometric method was often used tonitrate determination because this method was simple and
sensitive. Nitrate and nitrite can be investigated using the diazotization coupling reaction [10, 11, 14]. Nitrate was
changed into nitrite and formed azo dye under alkaline conditions. The measurement of nitrate can be obtained using

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AIP Conf. Proc. 1911, 020012-1–020012-6; https://doi.org/10.1063/1.5016005
Published by AIP Publishing. 978-0-7354-1603-1/$30.00

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reaction nitrate with salicylic acid in sulfuric acid conditions [12]. Spectrophotometric analysis of nitrate and nitrite
was used vanadium(III) as an alternative amalgamated cadmium for nitrate reduced to nitrite [13].
Laboratory of Environmental Agency DI Yogyakarta is a laboratory that serves as the responsibility of
environmental quality control of the province of DI Yogyakarta which covers 5 districts of Bantul, Sleman,
GunungKidul Regency, KulonProgo Regency and Yogyakarta City. The focus of this laboratory work testing
surface water, water soil and waste water. Laboratories conducting routine testing should be able to demonstrate the
validity of the methods used. Test methods that have been standardized, verified in advance to ensure that the
method can be applied in laboratory testing by generating valid data. Verification is the process of proving the
suitability of the competence of testing, so that the results of a test can be accounted. This research verified the
spectrophotometric method to analyze nitrate in water samples using APHA 2012 Section 4500 NO3-B method.

MATERIAL AND METHOD

This study was using APHA 2012 Section 4500 NO3-B method and took place in the testing laboratory of
Environmental Agency of DI Yogyakarta Province. The materials used were HCl (Merck), nitrite ion standard (1000
mg NO3-/L), aquabidest and Reverse Osmosis (RO) water samples. Sample analysis used Shimadzu UV-1800 reader
spectrophotometer and followed 220 nm and 275 nm wavelength.
The verification parameters used were: linearity, method detection limit, level of quantitation, level of linearity,
accuracy and precision. Sample and standard solutions were added 1 mL HCl 1 M before measured using UV-Vis
spectrophotometer. Concentration of calibration standard solution were 0 mg/L; 10 mg/L; 20 mg/L; 30 mg/L; 40
mg/L and 50 mg/L. The calibration curve was made from the difference between absorbance data at 220 nm and
multiple of absorbance data at 275 nm.

RESULT AND DISCUSSION

Linearity
Linearity was obtained using calibration curve by nitrate concentration 10 to 50 mg/L. The calibration curve
was drawn by plotting the values of the absorbance do to nitrate ion concentration. The assessment of the
performance characteristics of the calibration curve shown on Table 1.The correlation coefficient showed the
relationship between nitrite ion concentration and its values of the absorbance was linear. The correlation coefficient
(R) and correlation coefficients (R2) were 0.9987 and 0.9973. This value of correlation coefficients (R2) evaluated in
the linearity that higher than 0.990 [2]. On the other hand, criteria linearity of response on analytical method if the
value of correlation coefficients (R2) is higher than 0.995 [9].

TABLE 1.The Performance Characteristic of the Calibration Function

Parameter Value
Coefficient of determination (R2) 0.9973
The slope of the calibration function, abs∙L∙mg-1 0.0533
The intercept, abs 0.0346
The residual standard deviation, mg∙L-1 0.0033
The standard deviation 0.0515

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 3

Absorbance
2

1
y = 0.053x + 0.035
R² = 0.997
0
0 10 20 30 40 50 60

C nitrate (mg/L)

FIGURE 1. Calibration Curve

Precision
Precision value is represented by deviation (SD) and relative standard deviation (%RSD) in repeatability.
Precision would be better if the value of coefficient of variation was smaller. The value of %RSD or coefficient of
variation increased with decreasing the analyte that analyzed [5]. The results of repeatability were demonstrated in
Table 2. The variation coefficient among seven independent repetitions at the same sample. The result of the value
of SD (standard deviation) and relative standard deviation (%RSD) were 0.0507 and 1.0886%. This study showed
that seven repetitions have the proximity of measurement results where was %RSD ≤ 2%.

TABLE 2. Study of Precision

Repetition Absorbance Concentration (mg/L) ( − )
1 0.2537 4.7198 0.0037
2 0.2544 4.7332 0.0055
3 0.2505 4.6583 3 × 10
4 0.2479 4.6084 0.0025
5 0.2483 4.6161 0.0018
6 0.2483 4.6161 0.0018
7 0.2506 4.6603 2 × 10
4.6589
0.0154
0.0507
%RSD 1.0886

Intermediate Precision
Intermediate precision was obtained with repeating examinations of test samples with different tools, timing
analysts, but in the same laboratory (Table 3). The value of ttablewas inscribed by determining degrees of freedom, df
= (n + n-2) at 95% confidence level, α/2 = 0.0525 where tstat (0.025; 12) was 2.56. The limit of acceptance of the
calculation wastcount<tstat with an indication that the test value for intermediate precision has no differences.

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 TABLE 3. Intermediate Precision Data
Absorbance Concentration (mg/L)
Repetition
Analysis 1 Analysis 2 Analysis 1 ( − ) Analyst 2 ( − )
1 0.2337 0.2353 4.3362 0.00293 4.3669 0.00479
2 0.2343 0.2302 4.3481 0.00436 4.2679 0.00089
3 0.2309 0.2287 4.2816 2.1E-07 4.2406 0.00326
4 0.2286 0.234 4.2372 0.00201 4.3413 0.0019
5 0.2289 0.2327 4.2440 0.00145 4.3174 0.00039
6 0.2289 0.2295 4.2440 0.00145 4.2560 0.00174
7 0.2310 0.2315 4.2833 1.5E-06 4.2935 1.7E-05
4.2821 4.2977
0.0122 0.01298
SD 0.0451 0.0465
SD2 0.0020 0.0022
df 12
0.1153
0.2532
2.5600

Accuracy
Accuracy was presented by recovery value from seven repetitions of spiked sample with standard solution
(Table 3). The recovery value from this study was 109.1907% and its acceptable recovery values vary from 85 to
115% [6]. This result indicated the method of nitrate analysis was accurate.Many factors that can affect the
accuracy, one of them was reaction condition where the formation of a stable reaction can affect the achievement of
the success of the reaction.

TABLE 4. Concentration Values contained in repeatability

Absorbance Concentration (mg/L)
Repetition %R
Sample Spiked sample Sample Spiked sample
1 0.0573 0.1725 0.9496 3.1611 110.5750
2 0.0573 0.1696 0.9496 3.1064 107.8400
3 0.0573 0.1697 0.9496 3.1080 107.9200
4 0.0573 0.1744 0.9496 3.1976 112.4000
5 0.0573 0.1726 0.9496 3.1628 110.6600
6 0.0573 0.1702 0.9496 3.1174 108.3900
7 0.0573 0.1683 0.9496 3.0806 106.5500
3.1334 109.1907

Method Detection Level (MDL) and Level of Quantitation (LoL)

Method Detection Level (MDL) was the analytical level determined according to the thorough method
testing stage to produce a signal with a 99% probability and the signal was different from the blank. Level of
Quantitation (LoQ) or minimum quantitation level (MQL) is an analytical level that produces larger signals from
blanks under routine laboratory conditions. Method Detection Level (MDL) was determined according to the

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response of standard deviation (SD) or value that produced a signal with 99% probability and the signal was
different from the blank (Equation 1).
MDL = 3.13 × SD (1)
LOQ = 10 × SD (2)

TABLE 5. Recovery values in accuracy

Concentration Spiked Sample
Repetition Absorbance ( − )
(mg/L)
1 0.1725 3.1611 0.0008
2 0.1696 3.1064 0.0007
3 0.1697 3.1080 0.0006
4 0.1744 3.1976 0.0041
5 0.1726 3.1628 0.0009
6 0.1702 3.1174 0.0003
7 0.1683 3.0806 0.0028
0.1711 3.1334 0.0015
0.04117
MDL 0.1294
LoQ 0.4117

The values of Method Detection Level (MDL) and Limit of Quantitation (LOQ) compared with the standard
concentration of standard nitrate solution.The values of MDL and LoQ were 0.1294 mg/L and 0.4117 mg/L. It was
the smallest concentration that does not cause bias in the calculation.The concentration of the samples detected by
this method were greater than the value of MDL and LOQ. Its indicated that value of standards and samples could
be accepted.

Level of Linearity (LoL)

Determination of LoL was done by making standard solution 10 mg/L and 50 mg/L with 10 times repetition.
The measurement results of Level of Linearity (LoL) can be seen in Table 5. This result was compared with Ftable
(0.01; 6; 6)= 8.466. The limit of acceptance of the calculation is Fcount<Ftable. It can be concluded that nitrate
concentration 10 to 50 mg/L was linear with level of confidence was 99% and value of LoL was 50 mg/L.
TABLE 6. Standard concentrations to obtained LoL
Repetition Absorbance of 10 Absorbance of 50
mg NO3-/L mg NO3-/L
1 0.5582 2.6297
2 0.5900 2.6391
3 0.5876 2.6486
4 0.5866 2.6474
5 0.5851 2.6452
6 0.5865 2.6432
7 0.5870 2.6363
8 0.5875 2.6398
9 0.5985 2.6409
10 0.5896 2.6206
SD 0.01035 0.00853
1.47119

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 CONCLUSION
This research was to verify the spectrophotometric method to analyze nitrate in water samples using APHA 2012
Section 4500 NO3-B method. The verification parameters used were: linearity, method detection limit, level of
quantitation, level of linearity, accuracy and precision. The tested performance criteria showed that the methodology
was verified under the laboratory conditions.

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