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Chapter 4: Liquid Chromatographic Determination of Nitrate and Nitrite
Ph. D Thesis: North Maharashtra University, Jalgaon, 2015 Page 78
4.1 Background
Recently developed fluorometric HPLC method involves the precolumn
derivatization of nitrite with 2, 3-diaminonapthalene and enzymatic conversion of nitrite
to nitrate [1]. Measurement of nitric oxide itself is complicated by its short half life and
would, therefore, require on-line measurement which is suitable for routine use for
analysis of biological samples. After an exhaustive investigation, it appears that HPLC,
which is a very sensitive, rapid and accurate method with low detection limits for nitrite
and nitrate, carries none of these disadvantages and nitrite and nitrate can be measured
directly. The chromatographic system developed in this study, after investigating
alternatives, readily resolved NO2- and NO3
-, with peaks being separated within a minute
[2]. There are some analytical methods that has been reported for simultaneous
estimation of nitrite and nitrate cited in references [3-15].
Lulla (1984) proposed a stability-indicating analytical method for the
simultaneous determination of nitrate, and if present, its reductive degradation product,
nitrite, in toothpastes. Nitrate and nitrite were extracted from the toothpaste using
distilled water and separated from other water-soluble excipients by two RP-8 columns
(250 mm L x 4 mm i.d.) using a mobile phase containing 0.2 % (w/v) sodium acetate and
2.5 % (v/v) glacial acetic acid in distilled water. A UV detector set at 313 nm was used
for quantitation. The method was applied to commercial toothpastes containing 5 %
potassium nitrate and yielded an average recovery of 100.1 % with a relative standard
deviation of 1.43 %. Average recovery of nitrate and nitrite from spiked samples was
100.6 % and 96.4 %, respectively. The minimum detectable concentration for nitrite was
50 micrograms/g of toothpaste [3]. Marcelo et al. (1996) developed a high-performance
liquid chromatographic method for the determination of nitrite and nitrate anions derived
from nitric oxide in biological fluids. After separation on a strong anion-exchange
column (Spherisorb SAX, 250 x 4.6 mm I.D., 5 µm), two on-line post-column reactions
occur. The first involves nitrate reduction to nitrite on a copper-plated cadmium-filled
column. In the second, the diazotization-coupling reaction between nitrite and the Griess
reagent (0.05 % naphtylethylendiamine dihydrochloride plus 0.5 % sulphanilamide in 5
% phosphoric acid) takes place, and the absorbance of the chromophore is read at 540
nm. Before injection into the chromatographic system, the samples were diluted and
Chapter 4: Liquid Chromatographic Determination of Nitrate and Nitrite
Ph. D Thesis: North Maharashtra University, Jalgaon, 2015 Page 79
submitted to suitable clean-up procedures (urine and cell culture supernatant samples are
passed through C-s cartridges and serum samples were subjected to deproteinization by
ultrafiltration through membranes with a molecular mass cut-off of 3000). The method
has a sensitivity of 30 p mol for both anions, as little as 0.05-0.1 ml sample volume is
required and linearity is observed up to 60 n mol for each anion tested [4]. Rizzo et al.
(1998) proposed a specific and simple method for the direct simultaneous detection of
extracellular nitrite (NO2-) and nitrate (NO3-) using high-performance liquid
chromatography separation with UV and electrochemical detection in series. These stable
end products of nitric oxide (NO) were determined in dialysis perfusate obtained through
in vivo brain microdialysis during and after experimental photo induced cerebral
ischemia in rats. The chromatographic conditions were optimized with a reversed-phase
column (250 x 46 mm) using 10 mM n-octylamine pH 6.0 as a mobile phase. Absorbance
was measured at 220 nm for NO3- detection; electrochemical detection was performed at
+0.7 V for NO2- evaluation. This assay system holds the advantages of in vivo
consecutive measurements, high precision, good reproducibility, technical simplicity, fast
response (about 7 min) and wide availability [5]. A simple and rapid fluorometric HPLC
method was developed by Hui Lia (2000) for determination of nitrite through its
derivatization with 2, 3-diaminonaphthalene (DAN). Nitrite, in standard solution, cell
culture medium, or biological samples, readily reacted with DAN under acidic conditions
to yield the highly fluorescent 2, 3-naphthotriazole (NAT). For analysis of nitrate, it was
converted to nitrite by nitrate reductase, followed by the derivatization of nitrite with
DAN to form NAT. NAT was separated on a 5-µm reversed-phase C8 column (150 x 4.6
mm I.D.) guarded by a 40 µm reversed-phase C18 column (50 x 4.6 mm I.D.), and eluted
with 15 mM sodium phosphate buffer (pH 7.5) containing 50 % methanol (flow-rate, 1.3
ml/min). Fluorescence was monitored with excitation at 375 nm and emission at 415 nm.
Mean retention time for NAT was 4.4 min. The fluorescence intensity of NAT was linear
with nitrite or nitrate concentrations ranging from 12.5 to 2000 nM in water, cell culture
media, plasma and urine. The detection limit for nitrite and nitrate was 10 p mol/ ml [6].
Erk (2001) reported ratio spectra derivative spectrophotometry and high-performance
liquid chromatography (HPLC) method for determination of metronidazole and
miconazole nitrate in ovules. The first method depends on ratio spectra first derivative
Chapter 4: Liquid Chromatographic Determination of Nitrate and Nitrite
Ph. D Thesis: North Maharashtra University, Jalgaon, 2015 Page 80
spectrophotometry by utilizing the linear relationship between substances concentration
and first derivative peak amplitude. The ratio first derivative amplitudes at 242.6
(1DD242.6), 274.2 (1DD274.2), 261.8 (1DD261.8), 273.5 (1DD273.5) and 281.5 (1DD281.5) nm
were selected for the assay of metronidazole and miconazole nitrate, respectively. The
second method is based on high-performance liquid chromatography on a reversed-phase
column using a mobile phase of methanol-water-phosphoric acid (30:70:0.20 v:v) (pH
2.8) with programmable detection at 220.0 nm. The minimum concentration detectable
by HPLC was 0.9 mg/ml for metronidazole and 0.3 mg/ml for miconazole nitrate and by
ratio derivative spectrophotometry 4.0 mg/ml for metronidazole and 0.5 mg/ml for
miconazole nitrate [7]. Krzek (2003) developed a HPLC method for determination of
propane-1,2,3-triyl trinitrate and impurities: (2RS)-3-hydroxypropane-1,2-diyl dinitrate
and 2-hydroxypropane-1,3-diyl dinitrate in ointment. For individual constituents R.S.D.
was ranged from 0.7 to 9.9 %, while recovery was 100.1 % for propane-1,2,3-triyl
trinitrate and 95.1-/99.0 % for impurities. It has been found that propane-1,2,3-triyl
trinitrate used in medicine in the form of ointment contains such impurities which can be
identified and quantified at relatively low concentrations of 70 ng/ml [8]. Shin-Shou
(2003) developed a simple, rapid, precise and sensitive high performance liquid
chromatography (HPLC) method using an UV detector was developed for the
determination of nitrate and nitrite amounts in vegetables. The optimal conditions were
found and applied using 0.01 M octylammonium orthophosphate of aqueous 30 % (v/v)
methanol of pH 7.0 for the mobile phase at flow rate of 0.8 ml/min. The total time for
one sample analysis was within 10 min. Recoveries of nitrate and nitrite was between
96.6 to 105.7 %. The calibration curves of nitrate and nitrite were extremely linear, where
both correlation coefficients were greater than 0.9990 in the range of 0.1~100.0 µg/ml.
For application, nitrate and nitrite amounts in 12 marketed vegetables were determined
by this HPLC method. The results showed nitrate and nitrite contents varied in a range of
225-4, 410 mg/kg and 5-200 mg/kg, respectively [9]. Yuegang (2006) proposed a
simple, fast, sensitive and accurate reversed-phase ion-pair HPLC method for
simultaneous determination of nitrite and nitrate in atmospheric liquids and lake waters
has been developed. Separations were accomplished in less than 10 min using a reversed-
phase C18 column (150 mm x 2.00 mm I.D., 5 µm particle size) with a mobile phase
Chapter 4: Liquid Chromatographic Determination of Nitrate and Nitrite
Ph. D Thesis: North Maharashtra University, Jalgaon, 2015 Page 81
containing 83 %, 3.0 mM ion-interaction reagent tetrabutylammonium hydroxide (TBA-
OH) and 2.0 mM sodium phosphate buffer at pH 3.9 and 17 % acetonitrile (flow rate, 0.4
ml/min). UV light absorption responses at 205 nm were linear over a wide concentration
range from 100 g/ml to the detection limits of 10 µg/l for nitrite and 5 µg/l nitrate.
Quantitation was carried out by the peak area method. The relative standard deviation for
the analysis of nitrite and nitrate was less than 3.0 %. This method was applied for the
simultaneous determination of nitrite and nitrate in dew, rain, snow and lake water
samples collected in southeast Massachusetts. Nitrate was found being present at 4.79-
5.99 µg/ml in dew, 1.20-2.63 µg/ml in rain, 0.32-0.60 µg/ml in snow and 0.12-0.23 µg/ml
in lake water [10].
Angel (2008) has been developed and validated a reversed-phase high
performance liquid chromatographic (RP-HPLC) method for determination of econazole
nitrate, preservatives (methylparaben and propylparaben) and its main impurities (4-
chlorobenzyl alcohol and alpha-(2,4-dichlorophenyl)-1,H-imidazole-1-ethanol) in cream
formulations. Separation was achieved on a column Bondclone C18 (300 mm x 3.9 mm
I.D., 10 µm particle size) using a gradient method with mobile phase composed of
methanol and water. The flow rate was 1.4 ml min−1, temperature of the column was 25
C and the detection was made at 220 nm. Miconazole nitrate was used as an internal
standard. The total run time was less than 15 min. The analytical curves presented
coefficient of correlation upper to 0.99 and detection and quantitation limits were
calculated for all molecules. Excellent accuracy and precision were obtained for
econazole nitrate. Recoveries varied from 97.9 to 102.3 % and intra and inter-day
precisions, calculated as relative standard deviation (R.S.D.), were lower than 2.2 %.
Specificity, robustness and assay for econazole nitrate were also determined [11]. A rapid
and cost-effective RP-HPLC method with diode array detector was optimized and
validated by Ferreira (2008) for quantification of nitrites and nitrates in ham. The
chromatographic separation was achieved using a HyPurity C18, 5 µm chromatographic
column and gradient elution with 0.01 M n-octylamine and 5 mM tetrabutylammonium
hydrogenosulphate to pH 6.5. The determinations were performed in the linear range of
0.0125-10.0 mg/l for nitrite and 0.0300-12.5 mg/l for nitrate. The detection limits for
nitrite and nitrate were 0.019 and 0.050 mg/kg, respectively. The reliability of the method
Chapter 4: Liquid Chromatographic Determination of Nitrate and Nitrite
Ph. D Thesis: North Maharashtra University, Jalgaon, 2015 Page 82
in terms of precision and accuracy was evaluated. Coefficients of variation lower than
2.89 and 5.47 % were obtained for nitrite and nitrate, respectively. Recoveries of residual
nitrite and nitrate ranged between 93.6 and 104.3 %, respectively. Analysis of cooked and
dried ham samples was performed, and the results obtained were in agreement with
reference procedures [12]. Kodamatani (2009) developed a simple, sensitive and selective
method for the simultaneous determination of nitrite and nitrate in water samples. The
method was based on ion-exchange separation, online photochemical reaction, and
luminol chemiluminescence detection. The separation of nitrite and nitrate was achieved
using an anion-exchange column with a 20 mM borate buffer (pH 10.0). After the
separation, these ions were converted to peroxynitrite by online UV irradiation using a
low-pressure mercury lamp and then mixed with a luminol solution prepared with
carbonate buffer (pH 10.0). The calibration graphs of the nitrite and nitrate were linear in
the range from 2.0x10-9 to 2.5x 10-6 and 2.0x10-8 to 2.5x10-5 M, respectively. Since the
sensitivity of nitrite was about 10 times higher than that of nitrate, the simultaneous
determination of nitrite and nitrate in the water samples could be efficiently achieved.
This method was successfully applied to various water samples, river water, pond water,
rain water, commercial mineral water, and tap water with only filtration and dilution steps
[13]. Yongtao Li et al. (2011) developed a new method for the analysis of nitrate and
nitrite in a variety of water matrices by using reversed-phase liquid
chromatography/electrospray ionization/mass spectrometry in the negative ion mode. For
this direct analysis method, nitrate and nitrite anions were well separated under the
optimized LC conditions, detected by monitoring m/z 62 and m/z 46 ions, and quantitated
by using an isotope dilution technique that utilized the isotopically labeled analogs. The
method detection limits, based on seven reagent water replicates fortified at 0.01 mg/L
nitrate and 0.1 mg/L nitrite, were 0.001 mg/L for nitrate and 0.012-0.014 mg/L for nitrite.
For the analysis of nitrate and nitrite in drinking water, surface water, and groundwater
samples, the obtained results were generally consistent with those obtained from the
reference methods. The mean recoveries from the replicate matrix spikes were 92-123 %
for nitrate with an RSD of 0.6-7.7 % and 105-113 % for nitrite with an RSD of 0.3-1.8 %
[14]. Anguo Wu (2013) developed a simple, cost-effective and accurate HPLC method
for the determination of nitrite and nitrate. On the basis of the reaction that nitrite is
Chapter 4: Liquid Chromatographic Determination of Nitrate and Nitrite
Ph. D Thesis: North Maharashtra University, Jalgaon, 2015 Page 83
oxidized rapidly to nitrate with the addition of acidic potassium permanganate, the
determination of nitrite and nitrate was achieved by the following strategy: each sample
was injected twice for HPLC analysis, i.e. the first injection was to measure nitrate, and
the second injection was to measure total nitrate including initial nitrate and the nitrate
from the conversion of nitrite with the addition of acid potassium permanganate in the
sample. The amount of nitrite can be calculated as difference between injections 2 and 1.
The HPLC separation was performed on a reversed phase C18 column for 15 min. The
mobile phase consisted of methanol-water (2:98 by volume); the water in the mobile
phase contained 0.60 mM phosphate salt (potassium dihydrogen and disodium hydrogen
phosphate) and 2.5 mM tetrabutylammonium perchlorate (TBAP). The UV wavelength
was set at 210 nm. Additionally, systemic investigation of the effects of the concentration
of phosphate salt and TBAP in the mobile phase, the pH of the mobile phase, and the
amount of acidic potassium permanganate added to the sample on the separation efficacy
was carried out. The results showed that the limits of detection (LOD) and the limit of
Quantitation (LOQ) were 0.075 and 0.25 µM for nitrate (containing the oxidized nitrite),
respectively. The linear range was 1-800 µM [15].
In this work, a new reversed-phase HPLC method has been developed
for the analysis of nitrite and nitrate in various samples. The method uses RP-HPLC
based on isocratic mode analogs to nitrite and nitrate. The nitrate and nitrite anions were
well separated by the selected HPLC column under a moderate pH condition.
Derivatization was not required for this method. The study was focused on the
selection and optimization of reversed-phase HPLC conditions, the demonstration of
method performance (sensitivity, accuracy and precision) and the investigation of
interferences from common inorganic anions.
Chapter 4: Liquid Chromatographic Determination of Nitrate and Nitrite
Ph. D Thesis: North Maharashtra University, Jalgaon, 2015 Page 84
4.2 Material and Methods
4.2.1 Equipments
Table 4.1: Instrumentation used in RP-HPLC Method Development
Sr. No. Name of Instrument Make Model
1 HPLC System Shimadzu, Japan Software–LC Solution Pump-LC 2010 CHT Detector- UV
2 Analytical Balance Shimadzu, Japan AUY 120
3 Digital pH Meter Electronic Corporation Ltd., India
pH 5651
4 Ultrasonicator Servewell Instrument, India RC System MU-1700
5 Degasser Tarsons, India Rockyvac-300
6 Milli-Q water purification system
Bedford, MA, USA
Millipore
4.2.2 Reagents and Materials
A. Standards 1. Potassium nitrate (Merck, India)
2. Sodium nitrite (Merck, India)
3. Sodium chloride, sodium sulfate, potassium phosphate (monobasic), and
sodium carbonate (Fisher Scientific, India)
4. Milli-Q water with 18.2 MΩ-cm resistances (Millipore water system)
B. Real Samples
1. Water samples- Tap, Ground and Surface water from local wells and river
2. Vegetable samples- Spinach, Coriander leaves, Beet, Radish leaves, Sorrel,
Cabbage, Tomato and Mint from local market
3. Soil sample- From local farm
4. Pharmaceutical preparation- Isosobidinitrate tablets from Nichoals Piramal
C. Chemicals and Reagents
1. Methanol (HPLC grade), Merck (India)
Chapter 4: Liquid Chromatographic Determination of Nitrate and Nitrite
Ph. D Thesis: North Maharashtra University, Jalgaon, 2015 Page 85
2. Potassium dihydrogen Phosphate AR Grade, Qualigens (India)
3. Orthophosphoric acid AR Grade, Qualigens (India)
4.3 Results and Discussion
4.3.1 Selection of Common Solvent (Diluent)
HPLC grade water was selected as common solvent for preparation of stock
solution and developing spectral characteristics of nitrate and nitrite, further dilutions from
stock solutions were made in mobile phase of aqueous HPLC grade methanol.
4.3.2 Determination of λmax of Nitrate and Nitrite
Preparation of Standard Stock solution: Weighed accurately 0.01500 gm sodium
nitrite and 0.016 gm potassium nitrate working standard in a 10.0 ml volumetric flask and
dissolved by sonication in sufficient mobile phase then make up the volume by mobile
phase. Dilute 1.0 ml of this solution to 10.0 ml with mobile phase i.e. methanol.
The aliquot portion of standard stock solutions of nitrate and nitrite were diluted
appropriately with aqueous methanol to obtain concentration 10 ppm of each analyte. The
solutions were scanned in the range of 200-400 nm. The absorbance spectrum of nitrate
and nitrite is shown in Figure 4.1 & Figure 4.2, respectively whereas the overlain
spectra of both the ions in mixture are shown in Figure 4.3.
It is observed as the wavelengths of maximum absorption, λmax, for nitrate and
nitrite were 230 and 233 nm, respectively. From the overlain spectra, the wavelength
selected for simultaneous estimation of nitrate and nitrite was 222 nm as an isoabsorptive
point for nitrate and nitrite ions.
Figure 4.1; Absorbance Spectrum of Nitrate on UV Detector
Chapter 4: Liquid Chromatographic Determination of Nitrate and Nitrite
Ph. D Thesis: North Maharashtra University, Jalgaon, 2015 Page 86
Figure 4.2: Absorbance Spectrum of Nitrite on UV Detector
Figure 4.3: Overlain Spectra of Nitrate and Nitrite
4.3.3 Optimization of Chromatographic Conditions
The mobile phase was allowed to equilibrate with stationary phase until steady
baseline was obtained. The standard solution containing mixture of nitrate and nitrite was
run and different individual solvents as well as combinations of solvents have been tried to
get a good separation and stable peak. Each time mobile phase was filtered through 0.45
µm filter membrane. Based on sample solubility and stability, various mobile phase
compositions were evaluated to achieve acceptable separation using selected
chromatographic conditions.
Chapter 4: Liquid Chromatographic Determination of Nitrate and Nitrite
Ph. D Thesis: North Maharashtra University, Jalgaon, 2015 Page 87
The various conditions of mobile phases, flow rate and pH buffer tried are
represented in Table 4.2 and chromatograms obtained in Trial 1, 2 & 3 are shown in
Figure 4.4, 4.5 & 4.6, respectively..
Table 4.2: Trials for Variable Chromatographic Parameters
Chromatographic
Parameters
Trial 1 Trial 2 Trial 3
Column C13 C13 C13
Wavelength 222 nm 222 nm 222 nm
Flow rate 0.4 ml/min 0.4 ml/min 0.7 ml/min
Injection volume 20.0 µl 20.0 µl 20.0 µl
Column oven Temperature
25 0C 25 0C 25 0C
Run Time less than 10 minutes less than 10 minutes
less than 10 minutes
Mobile Phase Methanol:Water
( 30:70)
Methanol:Water
( 20:80)
Methanol:Buffer
( 20:80)
pH buffer
not used not used Potassium dihydrogen phosphate ( pH=3.0)
Figure 4.4: Chromatogram of Nitrate and Nitrite for Trial 1
Chapter 4: Liquid Chromatographic Determination of Nitrate and Nitrite
Ph. D Thesis: North Maharashtra University, Jalgaon, 2015 Page 88
Figure 4.5: Chromatogram of Nitrate and Nitrite for Trial 2
.
Figure 4.6: Chromatogram of Nitrate and Nitrite for Trial 3
Chromatogram of Trial 1 shows very poor resolution, Trial 2 also shows less
resolution between nitrate and nitrite; hence those method parameters were not suitable.
The chromatographic conditions of Trial 3 were established by trial and error and were
kept constant throughout the method because proper peak shape, resolution and system
suitability was observed within limits.
4.3.4 Optimization of the Mobile phase, pH and Flow Rate
The various aqueous methanol concentrations (10, 20, 25 and 30 %, v/v) and
different pH values (2.0, 2.5, 3.0 and 3.5) of mobile phase solutions at various flow rates
(0.4, 0.7 and 1.0 ml/min) were tested on running HPLC chromatograms. As shown in
Figure 4.7 the 20 % methanol gives the proper resolution. On 10 % methanol the
resolution is achieved but the retention time (RT) is long and 25 and 30 % methanol
concentrations in mobile phase gave less resolution. Figure 4.8 show that 3.7 is the
Chapter 4: Liquid Chromatographic Determination of Nitrate and Nitrite
Ph. D Thesis: North Maharashtra University, Jalgaon, 2015 Page 89
optimum pH for the best resolution in RT. This pH is intermediate between the pKa
values of both analytes. In the case of flow rate, Figure 4.9 shows that the 0.7 ml/min is
the preferred flow rate. On 0.4 ml/min flow rate the RT is too long and at 1.0 ml/min
flow rate, the resolution is not achieved. So the 20 % methanol at pH 3.0 and 0.7 ml/min
flow rate is considered as set of optimized chromatographic conditions. Figure 4.10
represents the resolved peak of nitrite and nitrate at optimized chromatographic
conditions.
Figure 4.7: Effect of Variation of Methanol Concentration in Mobile Phase
Figure 4.8: Effect of Variations of pH of Mobile Phase
0
2
4
6
8
10
0 10 20 30 40
Ret
enti
on
Tim
e
Methanol Concentration in %
NitriteNitrate
012345678
1.5 2 2.5 3 3.5 4
Ret
enti
on
Tim
e
pH of mobile phase
NitriteNitrate
Chapter 4: Liquid Chromatographic Determination of Nitrate and Nitrite
Ph. D Thesis: North Maharashtra University, Jalgaon, 2015 Page 90
Figure 4.9: Effect of Variation of Flow Rate of Mobile Phase
Figure 4.10: Separation of Nitrite and Nitrate in selected mobile phase showing retention time for Nitrite (1ppm) and Nitrate (10ppm)
4.3.5 Preparation of Calibration Curve
i) Preparation of Nitrite standard stock solution (1000 ppm) : An accurately weighed
quantity of sodium nitrite 0.015 gm was transferred to the 10.0 ml volumetric flask
and dissolved in 20 % methanol by sonication. The volume was made up to the mark
with 20 % methanol.
ii) Preparation of Nitrate standard stock solution (1000 ppm) : An accurately weighed
quantity of potassium nitrate 0.016 gm was transferred to the 10.0 ml volumetric
flask and dissolved in 20 % methanol by sonication. The volume was made up to the
mark with 20 % methanol.
0
2
4
6
8
10
0 0.2 0.4 0.6 0.8 1 1.2
Ret
enti
on
Tim
e
Flow rate of mobile phase (ml/min)
Nitrite
Nitrate
Chapter 4: Liquid Chromatographic Determination of Nitrate and Nitrite
Ph. D Thesis: North Maharashtra University, Jalgaon, 2015 Page 91
The aliquot portions of standard stock solutions of nitrite and nitrate were diluted
appropriately with 20 % methanol to obtain the series of concentrations from 1 to
100 ppm of nitrate and 0.1 to 10 ppm of nitrite.
ii) Procedure: The mobile phase was allowed to equilibrate with the stationary phase
until steady baseline was obtained. The series of concentrations from 1-100 ppm of
nitrate and 0.1-10 ppm of nitrite solutions were injected and peak area was recorded.
The standard calibration curve for nitrite and nitrate are represented in Figure 4.11
& 4.12, respectively.
Figure 4.11: Calibration Curve for Nitrite on HPLC Instrument
Figure 4.12: Calibration Curve for Nitrate on HPLC Instrument.
y = 1703.x + 69.43R² = 0.999
02000400060008000
1000012000140001600018000
0 2 4 6 8 10 12
Pea
k A
rea
NO2- concentration, ppm
y = 376.6x - 71.17R² = 0.999
0
5000
10000
15000
20000
25000
30000
35000
40000
0 50 100 150
Pea
k A
rea
NO3- concentration, ppm
Chapter 4: Liquid Chromatographic Determination of Nitrate and Nitrite
Ph. D Thesis: North Maharashtra University, Jalgaon, 2015 Page 92
4.3.6 System Suitability Test
System suitability is used to verify, whether the resolution and reproducibility of
the chromatographic system are adequate for analysis to be carried out. It is performed to
ensure that the system is operating properly and ready to deliver results with acceptable
accuracy and precision. The tests were performed by collecting data from five replicate
injections of standard solutions. The Filtered mobile phase was allowed to equilibrate with
the stationary phase until steady baseline was obtained. A 20 µl standard nitrate and nitrite
solutions were injected which were made in five replicates and the system suitability
parameters were recorded as shown in Table 4.3 & 4.4 for nitrate & nitrite, respectively.
System suitability parameters criteria:
a. % R.S.D. of the area of analyte peakf in standard chromatogram should not be more
than 2 %.
b. Theoretical plates of analyte peaks in standard chromatogram should not be less than
2000.
c. Tailing factor (Asymmetry) of analyte peaks in standard chromatogram should be less
than 2.0.
d. Retention time (Rt) of analyte peaks in standard chromatogram should not be more than
1 %.
Table 4.3: System Suitability Test Results for Nitrate
Sr. No. Area
Reproducibility
Retention
Time
Tailing Factor (Asymmetry)
Theoretical
plates
1 3693.463 6.645 2.366 94054.844
2 3691.292 6.614 2.452 94136.76
3 3693.721 6.628 2.379 94095.27
4 3694.284 6.638 2.408 94183.18
5 3692.583 6.647 2.386 94068.25
Mean 3693.0686 6.6344 2.3982 94107.6608
SD 1.167 0.014 0.034 52.551
% RSD 0.0316 0.2052 1.4055 0.0558
Limit NMT 2.0 % NMT NMT 2 % NLT 2000
Chapter 4: Liquid Chromatographic Determination of Nitrate and Nitrite
Ph. D Thesis: North Maharashtra University, Jalgaon, 2015 Page 93
Table 4.4: System Suitability Test Results for Nitrite
Sr. No. Area
Reproducibility
Retention
Time
Tailing Factor (Asymmetry)
Theoretical
plates
1 1776.86 5.189 1.36 129055
2 1775.79 5.213 1.372 129067
3 1778.63 5.227 1.415 129112
4 1784.91 5.311 1.365 129236
5 1778.41 5.262 1.378 129125.4
Mean 1778.92 5.2404 1.378 73.785
SD 3.545 0.047 0.022 0.0571
% RSD 0.1993 0.9063 1.5808 0.0571
Limit NMT 2.0 % NMT 1% NMT 2 % NLT 2000
4.3.7 Application of Proposed Method on Real Samples
4.3.7.1 Preparation of Mobile Phase and Standard Solutions
The optimal conditions of the mobile phase (20 % aqueous methanol, pH 3.0 and
flow rate 0.7 ml/min) were used in the experiment. Other chromatographic conditions
were those used in trial chromatogram run. Standard solution diluted to a series of
concentrations containing 0.1, 5, 10, 50, 100 ppm of nitrate and nitrite were prepared and
stored at 4 oC for use. The solutions were freshly prepared every seven days. The
standard curve and calculated correlation coefficients represents linearity within the
tested range of concentrations.
4.3.7.2 Sample Preparation for Analysis
All vegetable samples including coriander leaves, radish leaves, spinach leaves
and cabbage were bought from the local morning market. The vegetable samples were
carefully rinsed with tap water and deionized water and were divided into smaller pieces.
They were dried at 105 °C for 24 h to constant weight. The dried samples were
pulverized and sieved to 60 mesh. The resultant powders were stored in desiccators until
analysis. Dry and powdered samples of vegetables weighing to 0.1 g were added to a
beaker containing approximately 15 ml water. The sample solutions were stirred and
heated in boiling water bath for 8 h and transferred to 25 ml volumetric flasks. The
Chapter 4: Liquid Chromatographic Determination of Nitrate and Nitrite
Ph. D Thesis: North Maharashtra University, Jalgaon, 2015 Page 94
sample solutions were shaken for 30 min in supersonic wave, and diluted to a final
volume of 25 ml with deionized water. All samples were filtered through membrane filter
before use.
4.3.7.3 HPLC Analysis
The mobile phase is filtered through 0.45 µm membrane filter and sonicated for
degassing in an ultrasonic bath and then allowed to pass through the HPLC column until a
stable baseline signal was observed. The flow rate was 0.7 ml/min and the detecting UV
wavelength was 222 nm. Equal volumes (20.0 µL) of standard and sample solutions were
injected separately after equilibrium of mobile phase with stationary phase. The
chromatograms were recorded at optimized conditions and the response i.e. peak area of
major peaks was measured. The content of nitrate and nitrite were calculated by
comparing a sample peak with that of standard.
The injections of the standard solutions gave reproducible retention times and
peak areas with relative standard deviation (RSD) below 2.0. The peaks of the sample
were identified by comparison to the respective peaks of the standards. The amounts of
nitrate and nitrite in the test solution were calculated from the peak areas by using linear
regression equations of nitrate and nitrite standard curves. If the curve of the peak areas
was larger than that of the maximum amount from the standard curve, the test solution
was diluted to suitable concentrations.
4.3.7.4 Determination of Nitrate and Nitrite Contents in Vegetables and Water
The results for nitrate and nitrite analysis of the eight selected vegetables from
local market showed that the nitrite and nitrate contents varied significantly in the range
of 0-9.8 ppm and 57-1540 ppm, respectively (Table 4.5). The wide ranges and large
standard variations in nitrate and nitrite levels for the same vegetables purchased from
different places and periods due to the fact that nitrate levels in vegetable plants are
highly sensitive to inherent and environmental variables such as species, maturity,
fertilizer application and storage temperature. The nitrate amounts in vegetables have
reached to hazardous levels.
Chapter 4: Liquid Chromatographic Determination of Nitrate and Nitrite
Ph. D Thesis: North Maharashtra University, Jalgaon, 2015 Page 95
Table 4.5: Nitrite and Nitrate Content of Vegetables and Water Samples (n= 3)
Sample Nitrite content
ppm±SD Nitrate content
ppm±SD Coriander leaves 5.22±0.08 114.4±0.51
Radish leaves 7.11±0.07 220.7±0.58
Beet 9.83±0.15 1200.7±1.53
Spinach 9.40±0.10 1539.7±0.58
Sorrel 2.13±0.06 135.3±1.53
Cabbage 3.77±0.42 59.6±1.44
Tomato 3.90±0.10 57.8±0.76
Mint 6.0±0.10 164±1.0
Tap Water 0.21±0.01 2.97±0.15
Ground water 0.4±0.02 5.0±0.17
4.4 Validation of Developed RP-HPLC Method Parameters
4.4.1 Accuracy
The recoveries of nitrite and nitrate in the study are shown in Table 4.6 and 4.7.
The recoveries of nitrite for five concentrations (0.1, 0.5, 1, 5 and 10 ppm) into vegetable
samples were in the range of 96.4 to 100.3 % and nitrate for five concentrations (1, 5, 10,
50 and 100 ppm) into vegetable samples were in the range of 98.3 to 100.3 %. The
average of recovery is determined and recovery precision values were characterized by
the relative standard deviation (RSD %). The average recoveries of nitrite and nitrate
were 98.73% and 99.03 %, indicating that the method is quite accurate.
Table 4.6: Recovery Results of Nitrite Analysis for HPLC Method (n=3)
Spiked level
ppm % Average
Recovery SD
% RSD
0.1 96.47 0.9 0.8 0.5 99.43 1.0 1.0
1 97.11 0.7 0.7
5 100.26 0.4 0.4
10 100.37 0.9 0.9
Average 98.73 0.8
Chapter 4: Liquid Chromatographic Determination of Nitrate and Nitrite
Ph. D Thesis: North Maharashtra University, Jalgaon, 2015 Page 96
Table 4.7: Recovery Results of Nitrate Analysis for HPLC Method (n=3)
Spiked level
ppm % Average
Recovery
SD
% RSD
1 98.30 1.2 1.2
5 99.11 1.1 1.1
10 100.30 0.6 0.6
50 98.90 0.8 0.8
100 98.54 0.9 0.9
Average 99.03 0.9
4.4.2 Precision
Reproducibility tests, Intra-day (running 3 times on the same day), and inter-day
tests (running 3 times within successive 7 days with at least 24 h as intervals) were
conducted. The reproducibility precision values were characterized by the relative
standard deviation (RSD %). The intra-day and inter-day precision showed that the
results were within acceptable limit i.e. % R.S.D. below 2.0 indicating that the method is
reproducible. Intra-day and Inter-day Precision are represented in Table 4.8 & 4.9.
Table 4.8: Intra-day and Inter-day Precision Data of Nitrite for HPLC Method
Conc. of Nitrite ppm
% RSD
Intra-day Inter-day
0.1 1.2 1.5
0.5 0.9 1.2
1 0.6 0.2
5 0.4 0.4
10 1.3 1.2
Chapter 4: Liquid Chromatographic Determination of Nitrate and Nitrite
Ph. D Thesis: North Maharashtra University, Jalgaon, 2015 Page 97
Table 4.9: Intra-day and Inter-day Precision Data of Nitrate for HPLC Method
Conc. of Nitrate ppm
% RSD
Intra-day Inter-day 1 1.1 1.4
5 0.3 0.8
10 0.8 0.9
50 0.2 0.3
100 0.1 0.2
4.4.3 Specificity
In order to remove potentially interfering substances, the sample can be further
cleaned on an SPE anion exchange cartridge. The filtrate can then be used directly for
HPLC analysis. Fluoride, chloride, sulphate and sulfite are optically transparent, do not
interfere the UV absorbance measurement and thus make the simultaneous determination
of nitrite and nitrate possible [ 35]. Thus the same chromatogram for real sample analysis
was obtained as shown in Figure 4.10.
4.4.4 Limit of Detection(LOD) and Limit of Quantitation(LOQ)
Nitrite and nitrate sample solution was subjected to Limit of Detection(LOD) and
Limit of Quantitation(LOQ) studies,results are given in Table 4.10.
Table 4.10: LOD and LOQ of Nitrite and Nitrate for HPLC Method
Sample LOD (ppm) LOQ (ppm)
Nitrite 0.278 0.844
Nitrate 2.592 7.853
4.4.5 Linearity and Range
To show linerity and range of sample solution, the working range of analyte was
set between 0.1 to 10 ppm for nitrite and 1 to 100 ppm for nitrate. Figure 4.11 & 4.12
provided the standard curves of nitrite and nitrate. Linearity was obtained over the tested
concentration range of 0.1, 0.5, 1, 5 and 10 and 1, 5, 10, 50 and 100 ppm of nitrite and
nitrate, respectively. The linear regression equations of nitrite and nitrate standard curves
were calculated as y = 1703x + 69.43 (R2 = 0.9990) and y = 376.6x + 71.17 (R2 = 0.9990)
Chapter 4: Liquid Chromatographic Determination of Nitrate and Nitrite
Ph. D Thesis: North Maharashtra University, Jalgaon, 2015 Page 98
respectively. y is the value of peak area and x is the value of various concentrations of
standard solutions The correlation coefficients were both greater than 0.999, which
showed a very good linearity within the range receptive to nitrite and nitrate.
4.4.6 Robustness
The real sample of nitrite was analyzed using proposed method after a deliberate
change in detection wavelength for estimation by ±2 nm.
Table 4.11: Robustness Study Data of Nitrite and Nitrate for HPLC Method
Sr. No. Change in
wavelength
(±2 nm)
% Estimation
Nitrite Nitrate
1 220 98.66 98.91
2 222 98.75 99.01
3 224 99.09 98.65
Mean 98.83 98.86
±SD 0.2268 0.1858
% RSD 0.2295 0.188
The results indicates that the selected wavelength was unaffected by small variation in
the selected method parameters.
4.4.7 Ruggedness
Ruggedness study was carried out using only one parameter i.e. different analyst.
The result showed that the % RSD was in the range of 0.02-0.06 % and the result were
shown in Table 4.12. This study was signifies the ruggedness of the method under
varying conditions of its performance.
Table 4.12 Ruggedness Study Data of Nitrite and Nitrate for HPLC Method
Matrices Concentration
(ppm) Amount Found (%)
Analyst I Analyst II
Nitrite 1 99.86 97.75
Nitrate 10 96.51 96.82
Chapter 4: Liquid Chromatographic Determination of Nitrate and Nitrite
Ph. D Thesis: North Maharashtra University, Jalgaon, 2015 Page 99
4.5 Conclusion
1. The phosphate buffer of pH 3.7 and 20 % methanol at flow rate of 0.7 ml/min as
mobile phase is optimum for nitrite and nitrate analysis using HPLC method.
2. The wavelength of 222 nm is useful for simultaneous estimation of nitrate and nitrite
on UV detector.
3. Recoveries of nitrite and nitrate were better than 97 %.
4. A statistical analysis based on % R.S.D. indicates that the method is sufficiently
accurate as the values are less than 2 %.
5. The developed method is rapid, precise and sensitive and successfully applied for
determining nitrite and nitrate amounts in vegetable and water samples.
Chapter 4: Liquid Chromatographic Determination of Nitrate and Nitrite
Ph. D Thesis: North Maharashtra University, Jalgaon, 2015 Page 100
References:
1. H. Li, C. J. Meininger and G. Wu, Rapid determination of nitrite by reversed-phase
high-performance liquid chromatography with fluorescence detection, J.
Chromatography B, 746 (2000) 199-207.
2. S. A. Everett, M. F. Dennis, G. M. Tozer, V. E. Prise, P. Wardman and M. R. L.
Statford, Nitric oxide in biological fluids: analysis of nitrite and nitrate by high-
performance ion chromatography, J. Chromatography A, 706 (1995) 437-442.
3. H. Lulla, S. S. Chen and F. J. Sena, Simultaneous determination of nitrate and nitrite
in toothpastes by high-performance liquid chromatography, J. Pharm. Sci., 73 (7)
(1984) 1004-6.
4. M. N. Muscarfi and G.de Nucci, Simultaneous determination of nitrite and nitrate
anions in plasma, urine and cell culture supernatants by high-performance liquid
chromatography with post-column reactions, J. Chromatog. B, 686 (1996) 157-164.
5. V. Rizzo, L. Montalbetti, A. L. Rozza, W. Bolzani, C. Porta, G. Balduzzi, E. Scoglio
and R. Moratti, Nitrite/nitrate balance during photoinduced cerebral ischemia in the
rat determined by high-performance liquid chromatography with UV and
electrochemical detection, Journal of Chromatography A, 798 (1998) 103-108.
6. Hui Lia, C. J. Meiningerb and G. Wua, Rapid determination of nitrite by reversed-
phase high-performance liquid chromatography with fluorescence detection, Journal
of Chromatography B, 746 (2000) 199-207.
7. N. Erk and M. Levent Altun, Spectrophotometric resolution of metronidazole and
miconazole nitrate in ovules using ratio spectra derivative spectrophotometry and RP-
LC, Journal of Pharmaceutical and Biomedical Analysis, 25 (2001) 115-122.
8. J. Krzek, M. Moniczewska, G. Zabierowska-Slusarczyk, The HPLC determination of
propane-1,2,3-triyl trinitrite and impurities: (2RS)-3-hydroxypropane-1,2-diyl
dinitrate and 2-hydroxypropane-1,3-diyl dinitrate in ointment, Journal of
Pharmaceutical and Biomedical Analysis, 33 (2003) 403-409.
9. Shin-Shou Chou, Jen-Chien Chung and Deng-Fwu Hwang, A High Performance
Liquid Chromatography Method for Determining Nitrate and Nitrite Levels in
Vegetables, Journal of Food and Drug Analysis, 11 (3) (2003) 233-238.
10. Y. Zuo, Chengjun Wang and Thuan Van, Simultaneous determination of nitrite and
Chapter 4: Liquid Chromatographic Determination of Nitrate and Nitrite
Ph. D Thesis: North Maharashtra University, Jalgaon, 2015 Page 101
nitrate in dew, rain, snow and lake water samples by ion-pair high-performance liquid
chromatography, Talanta, 70 (2006) 281-285.
11. Angel A. Gaona-Galdos, Pedro López García, María S. Aurora-Prado, Maria Inês
Rocha Miritello Santoro and Érika Rosa Maria Kedor-Hackmann, Simultaneous
determination of econazole nitrate, main impurities and preservatives in cream
formulation by high performance liquid chromatography, Talanta, 77 (2008) 673-678.
12. I. Ferreira and S. Silva, Quantification of residual nitrite and nitrate in ham by
reverse-phase high performance liquid chromatography/diode array detector, Talanta,
74 (2008) 1598-1602.
13. H. Kodamatani, S. Yamazaki, Keiitsu Saito, Takashi Tomiyasu and Yu Komatsu,
Selective determination method for measurement of nitrite and nitrate in water
samples using HPLC with post-column photochemical reaction and
chemiluminescence detection, J. Chromatography A, 1216 (2009) 3163-3167
14. Y. Li, J. S. Whitaker and Christina L. McCarty, Reversed-phase liquid
chromatography/electrospray ionization/ mass spectrometry with isotope
dilution for the analysis of nitrate and nitrite in water, J. Chromatography A,
1218 (2011) 476-483.
15. A. Wu, T. Duan, D. Tang, Y. Xu, L. Feng, Z. Zhu, R. Wang and Q. Zhu,
Determination of Nitric Oxide-Derived Nitrite and Nitrate in Biological Samples by
HPLC Coupled to Nitrite Oxidation, Chromatographia, 76 (2013) 1649-1655.