Spectrophotometric Determination of Nitrate in Vegetables Using Phenol Gaya, U I Alimi, S
Spectrophotometric Determination of Nitrate in Vegetables Using Phenol Gaya, U I Alimi, S
Spectrophotometric Determination of Nitrate in Vegetables Using Phenol Gaya, U I Alimi, S
March, 2006
All rights reserved www.bioline.org.br/ja Vol. 10 (1) 79 - 82
ABSTRACT: A rapid and sensitive spectrophotometric method for the determination of nitrate in vegetables
is described. The method is based on the measurement of the absorbance of yellow sodium nitrophenoxide formed
via the reaction of phenol with the vegetable-based nitrate in presence of sulphuric acid. The analytical
concentration of the acid has marked effect on the nitrate determined. The colour development was rapid and
remained stable overnight. Analysis of six vegetable samples containing nitrate gave satisfactory mean recoveries
of 76 to 123% in 18 determinations. The proposed method is reproducible and sensitive to lower level
concentrations. @JASEM.
The high concentration of nitrogen in soil may lead to Toyoda et al. (1978) and a gas-liquid
the high nitrate levels in edible vegetables and toxic chromatographic method was developed. Basset et al.
levels of nitrite may be produced by microbial (1978) reported a titrimetric method for the nitrate
activity in the gastrointestinal tract of the consumer determination which could be erroneous if applied to
of such vegetables (Tanaka et al., 1982). Nitrate vegetables. Tanaka et al. (1982) reported a sensitive
containing vegetables are potential causes of and direct spectrophotometric method for the
methemoglobinemia, a disease which may occur in determination of nitrate in vegetables using 2-sec-
children older than 6 month (Sanchez-Echaniz et al., butyl phenol. The basis for the method is that 2-sec-
2001). Since nitrate-nitrogen is lost during digestion butylphenol reacts quantitatively with nitrate in
(Rowell, 1994), the determination of nitrate as acidic solution. Beijaars et al. (1994) reported a
nitrate-nitrogen via digestion becomes difficult. determination of nitrate in vegetables by continuous
Pfeiffer and Smith (1975) determined nitrate in baby flow procedure and subsequent reaction of the nitrate
food using ion selective electrode which showed a extracted with sulfanilamide and N-1-
good correlation with the AOAC xylenol method. naphthylethylenediamine to form a reddish-purple
Some of the methods employed are based on the azo dye which was measured colorimetrically at
nitration of bi-substituted phenol such as 2,4-xylenol, 530nm. This research aimed at developing a method
3,4-xylenol and 2,6-xylenol (Tanaka et al., 1982). for the determination of nitrate in vegetable products
Due to the involvement of distillation stage the 2,4- by using phenol as the active reagent. The principle is
xylenol method is time consuming (Tanaka et al., based on the nitration of phenol and the formation of
1982) and therefore unsuitable for routine food the corresponding sodium salt. The elementary
analysis. The 2,4-xylenol method was modified by processes involved are
NO2+
OH OH ......(3
NO2
NaOH
OH
ONa …… (4
NO2
NO2
Ultimately, 1 mol of sodium nitrophenoxide is provides a basis for the determination of nitrate as
produced from 1 mol of nitrate-nitrogen. Since nitrate-nitrogen. The reaction is quantitative and
sodium nitrophenoxide absorbs ultraviolet light it analogous to that reported by Tanaka et al. (1982).
*
Corresponding Author. E-mail: umargaya2000@yahoo.com
Spectrophotometric Determination of Nitrate… 80
0 RESULTS AND
0 5 10 15 20 DISCUSSION
The formation of sodium salt of
Nitrate µgml-1
nitrophenol through the reaction of
Fig 1a. Nitrate calibration curve vegetable- based nitrate with
phenol was quantitative and
provided a fundamental line for the
GAYA, U I; ALIMI, S
Spectrophotometric Determination of Nitrate… 81
determination of nitrate. The reaction was confirmed by the procedures Satisfactory recoveries of 76 to
of Boxer (1997) where upon addition of drops of concentrated HCl the 123% were obtained when the
yellow colour of the sodium phenoxide was discharged. The resulting sample of spinach, lettuce, water
alkaline extract was found to absorb at wavelength maximum of 407nm leaf, potato, tomato juice and onion
when measured with spectrophotometer. There was rapid colour were spiked with nitrate at level of
development and no measurable change in absorbance for 25minutes of 50 μgcm-3 (Table 1). Pfeiffer and
standing after full colour development (Fig 1). Smith (1975) used the similar
nitrate addition method to ensure
0.1
correct electrode response during
0.09 nitrate determination. BCS (1983)
0.08 had recoveries of up to 80 to 114%
0.07 of the nitrate added to six different
Absorbance
Fig 1 Effect of reaction time for nitration The graph indicated sharp rise in
the amount of nitrate detected as
the concentration of the acid is
The colour remained overnight. This is an indication of the stability of increased. Nitrate was not detected
the sodium nitrophenoxide. Evidently, the time of reaction does not when sulphuric acid concentration
have modifying effect in the analysis. Prior to the choice of toluene, was below 40%. The use of 98%
other solvents were tested as extractants but were found to be H2SO4 was therefore recommended
unsuitable. Benzene, xylene, ethanol and CCl4 formed emulsion during for the procedure. Nearly equal
the extraction process. Further, benzene extract appeared to be a dirty amounts of nitrate were determined
brown coagulation. Tanaka et al. (1982) found toluene suitable for successfully in aliquots of standard
extraction of phenoxide in the nitrate determination with 2-sec-butyl nitrate-nitrogen solutions at a level
phenol. Toyoda et al. (1978) used hexane to extract 6-nitro-2,4-xylenol of 10μgcm-3 by using different
in a modified 2,4-xylenol method for the determination of nitrate. phenol concentrations of 3, 5, 8, 10
and 15% (Fig 3). In the proposed
0.3 procedure 5% phenol was
recommended. There was no
0.25 significant difference in the amount
of nitrate determined with Na2CO3
0.2 concentration of 3, 5, 8, 10 and
Absorbance
GAYA, U I; ALIMI, S
Spectrophotometric Determination of Nitrate… 82
Table 1 Recoveries of nitrate added as KNO3 to certain vegetable suitability for analytical use. The
products at level of 50μgcm-3
reagents involved are common,
Sample Nitrate No of Mean cheap and suitable for routine
Found(μg) Determinations Recovery (%) analysis.
Spinach 308 + 14 3 123
Lettuce 296 + 9 3 118 Acknowledgement: The authors
Water 267 + 11 3 101
Leaf acknowledge the contribution of
Potato 236 + 8 3 94 Musa Beli of Central Laboratory
Tomato 185 + 6 3 74 Complex, Bayero University Kano.
Juice
Onion 189 + 6 3 76
REFERENCES
Bassett, J; Denney, RC; Jeffery,
GH; Mendham, J (1978)
Vogel’s Textbook of
0.3 Quantitative Inorganic
Analysis, ELBS and Longman,
0.25
London, pp686
Absorbance
0.2
BCS (1983) Colorimetric
0.15 Determination of Nitrate and
Nitrite in Foods, Laboratory
0.1 Procedure, LPFC-126, Health
Protection Branch
0.05 Laboratories, Bureau of
Chemical Safety, Ottawa,
0 Canada
0 5 10 15 20
Beijaars, PR; van Dijk, R; van der
% Phenol
Horst, GM (1994)
Fig 3. Influence of increasing phenol concentration on the Determination of Nitrate in
absorbance of nitrate Vegetables by Continuous
Flow: Interlaboratory Study, J.
Assoc. Off. Anal. Chem.
77(6):1522-9
0.3
0.25 Boxer, RJ (1997) Essentials of
Organic Chemistry. WCB /
Absorbance
GAYA, U I; ALIMI, S
Spectrophotometric Determination of Nitrate… 83
GAYA, U I; ALIMI, S