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Rapid Colorimetric Determination of Nitrate in Plant-Tissue by Nitration of

Salicylic-Acid

Article in Communications In Soil Science and Plant Analysis · January 1975

DOI: 10.1080/00103627509366547

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 6(1)', rr-30 (1975) CATAI,DO ET AL.
COMMIIN. soIL SCIENCE AND PLANT AMLYSIS'

ions interfere with the assay. McNanara et al.8 used a dis-

similatory nitrate reductase fron Fscherichia coli in an assay
which is extremely sensitive to nitrate-N; but the enzyne is
RAPIT,CoL0RIMETRICDETERMINATIoNoFNITRATEINPLANTTISSIJE

BY NITRATION OF SALICYLIC ACIDI

difficult to prepare, and the assay is tine-consuming. The
phenoldisulfonic acid nethod4 is versatile, but tedious.
KEY W0RDS: nitrosalicyllc acid, nitrate determination
We wanted a nethd that was rapid, free of interference
) fron other ions present in plant tissues, and suitable for
D.A. Cataldo, lll. llaroon, L.E. Schrader, and V'L' Youngs-

DePartment of AgronomY
samples with a wide range of nitrate-N conceiltrations. The
University of Wisconsin - l|adison procedure presented rneets these criteria.
lladison, llisconsin 53706
MATERIALS AND ME'IIIODS
ABSTRACT
Nitrate Extraction
An analysis is described for the raltid determination of Various plant parts from corn (Zea mays L.) and oats
nitrate-Ninplantextracts.Thecornplexforrnedhynitration (Avena sativa L.) that had been either freeze- or oven-dried
of salicylic acid under highly acidic conditions absorbs were ground to 40-mesh size. The ground sarnples vrere re-
maxirnally at 410 nn in solutions' Absorbance
basic (pH>I2) dried in an oven at 70oC and samples of 100 ng wete suspended
of the chromophore is directly proportional to the amount of in l0 ml of deionized water. The suspensions were incubated
nitrate-N present. Alrmoniun, nitrlte, and chloride ions do at for I hour. After mixing, the samples were centri-
45oC
not interfere. fuged at 5,000 X g for 15 ninlltes to sediment tissue residues.
The supcrnatants vere decanted and saved for analysis.

INTRODUCTION
in water or phosphate buffer3
Fresh tissue was homogenized

Nitrate in soils and plant tlssues has been deternined (l g fresh tissue per 6 rnl liquid). The honogenates were
II filtered and filtrates were centrifuged at 30,000 X g for l5
quantitatlve ly by Potentiornetric5 or spectrophotonetric4'6'7'
nethods. Sone of these nethods lack sensitivity, or other minutes. The supernatants nere decanted and saved for analysis.

7L

copynght @ l9?5 by Marccl Dekkcr, lnc. All Rights Rcserved. Neithcr this work nQr an)'ltrrt
may-beleproduccd or transmitted in any form or by any mcans, electronic or ntechanical. inclurling
photocopying, microfilmin3. and rccording, oI by 8ny information slorage and retlievsl systrnl'
sithout pcrmission in witin3 from thc publisher. 72
 DETERMIMTION OF NITMTE IN PLANT TISSUE
CATALDO ET AL.
Salicylic Acid Method for Nitrate Deternrination
by the phenoldisulfonic acid nethod' modified for sarnples
Aliquots (usually 0.2 nl of the extTacts nere pipetted
containing high chloride4. Aliquots (10 to 250 uf) also rere
into 50-rnl Erlenneyer flasks, and mixed thoroughly vith 0.8
assayed by the enzynatic rnethod8'
nt of 5t (w/v) salicylic acid in concentrated llrS0. (sA-H2s04).
P.ESULTS A\D DISCUSSION
After 20 rninutes at roon ternperature, 19 rnl of 2N Naoll were
The pmduct of the nitration reaction was scanned in a
added slowly with a Repipette (Lab Indrrstries, Inc., Berkeley,
Beckrnan Acta III spectrophotoneter; it showed naximal
absorn-
CA) to raise the pH above 12. Sanples were cooled to room
tion at 410 nrn (data not shown) ' Absorhance at 410 n|rl uas
temperature and absorhance at 410 nm was determined in a Gilford
linear frorn I to 60 ug NOI-N per aliquot, but aliquots con-
300-N spectrophotometer equipped with a rapid-sanpling cuvette
taining up to 600 uE can be assayed by further dilution of
(l-cn path length). Standards containing I to 60 ue NOI-H in
developed sanples with rater (Fig' i) ' llithout dilution' the
a 0.2 rnl aliquot were analyzed with each set of sarrPles. For

extracts from dried plant tissue, a hlank of 0.2 ml llr0 and

the normal reagents was sufficient, For fresh-tissue

norrnally 1.4

extracts, a separate blank was reqrrired for each sample because E

l.?
c
of pignentation in the extracts. The blank consisted of the o r.o
$
extract, 0.8 nl of concentrated H'SOO (without salicylic acid), o o8
(,
and 19 rnl of 2N NaOH, Nittate-N concentrations were expressed c
o o6
-c!
as ue N0l-N per g dry or fresh weight (ppn). L
o 04
l,l
.o
The SA-llrsOO reagent was nade fresh at least once each
o2
week and stored in a brown bottle. Nitrate standards were
o
stored at 4oC. 0 to 20 30 40 50 60
NO; -N (pgl2O ml)
Other Mcthods for Nitrate netermination
For conparison of the pmposed method with established
FIG. 1
nethods, aliquots (2 rnl) of sonre of the extTacts were assayed
Standard curve showing the linear response hetween absorbance
an<t NOi-N' N0i-N standards were added in 0'2 nI l'ater'
;---i Reaciion flasks contained I to 60 ug NOi-'\' -Ab-.
sorbance vas (leternined directly' l-----{
'Flask Reaction tlasKs
.ona"inua 50 to 600 pg llO!-N. contents ncre diluted
l:10 nith water before readlng the absorbancc'
73
74
 DETERMINATION OF NITMTE IN PLANT TISSUE CATAI.DO ET AI..

proposed rnethod is suitable for sanples containing SO0 to

increased proportionately, so that a ratio of at least 3:l
30,000 pg NO3-N per I dry wclsht (0.0S to 3r, No;-N). lltth (SA-ll2S04:llr0) is nnlntained, A snrall anount of water seems

dilution, the upper range can be extended lO-fold. For

deslrable as we consistently observed lower readings when no

sanrples containing less than 500 pg N0j-N per g dry weight, water was Present in the assay.
the tissue-extraction method (ratio of tissue:water) can be The prinary sources of interference in nitrate analyses
nodified to increase the concentration of NOl-N per aliquot, are chloride, nitrite, ions. With the pmposed
and anmonium

or a larger aliquot can be used with excess water being method, no interference was ohserved frorn nitrite or anmoniutn

evaporated before SA-!{2S04 is added. Color development is ions (Table l). Chloride inhibited at levels above 40 ug Cl
rapid and is stable for at least 48 hours in light or dark. per assay, but this level (equivalent to 2\ Cl- in plant tissue)
The effect of water (aliquot size) on color developrnent is far exceeds that normally present in plant tissrres. Addition of
shown in Fig, 2. i\laxirnal absorhance was obtained when sanples 200 ul of tloaglandrs nutrient solution (ninus N0l-N) to flasks
contained less than 0.3 ml water, r{hen nore water was present, containing 40 ug NOi-N caused no detectable interference (data
sensitivity and response linearity to N0;_N were lost. Aliquots
TABLE I
larger than 0.3 nl can be assayed if the arnount of SA-H,S0O is
Effcct of Adding Various Concentrations of Nitrite, Amnonium,
and Chloride to 40 ug N0!-N Before Assaying for Nitrate by
the Proposed Method.
E
c,
o o.5 Arnount of Additive in Presence of
Percent Recovery of N0!-N
\t Added Additives
(ue) Nitrite Anmonium Chloride
q,
(, o.4 0 100.1 t 0.1 100.2 r 0.1 99.8 I 0.2
c
o
o I 104.4 ! 0.6 102.0 t 0.8 98.9 r 3.0
o
o 4 101.5 r 0.3 100.0 t 0.1 104.0 t 3.2
4 o5
o. t o.2 0.3 o.4 l0 100.7 ! 0.2 99.7 ! 0.6 99.9 t 0.3
Atiquor (mt)
20 102.6 t 1.3 103.2 t 1.0 101.3 ! 0.4
FIG. 2
40 106.7 r 1.6 100.4 t 0.6 97.9 t 3.2
Fffect of various allquots of water on the nitration of sali_
cylic aci-rl ln srrlfuric acid, Nitrate (20 r:g N) was adderl 80 lll.l r 1.5 103.7 t 2.1 75.6 ! 0.5
to
a scries of flasks, evaporated to dryness, redissolved in vary_
ing amounts of rvater, and assayed by the iroposect nethod.

16
 CATALDO ET AL.
DATERHINATION OF NITMTE IN PLANT TISSUE

tained with the phenoldisulfonic acid method diffcred

someuhat
not shown). Thus, the nethod is suitable for nitrate-uptake
from values obtained uith the other nrethods'
studies, in which disappearance of N0;-N fron nutrient solutions
Nitrate was added to fresh extracts from oat and corn
is a neasure of nitrate uptake by plants'
leaves. Recovery of NOi-N by the proposed rnethod ranged from
The proposed nethod was conpared to the phenoldisulfonic
97. I to l}l.2% (data not shown) '
acid4 and dissinilatory nitrate assays (Table 2) '
""d"tu'"8 Nitrationofsalicylicacid(2-hydroxybenzoicacid)should
Generally, the phenoldisulfonic acid nrethod is adequate for
occur principally at carbon five, which is Para
to the hydroxyl
sanples containing rnore than 1000 ppm N0;-N" The enzvme
method
group. The hydroxy! group is ortho- or Dara-directing' while
is extrernely sensitive (ca' 0.1 ug Hol-N per l0 vl aliquot);
the carboxyl group is primarily meta-directingg' This
suggests
however, the enzyne is difficult to prepare and the assay
re-
that both functional Sroups should promote nitration of the
quires several hours. 'lhe proposedsalicylic acid method and
salicylic acid at carbon five' Stalctrp and Williansl0 reported
the enzyne nethod gave similar values (i'able 2)' Values ob-
that the nitro are essentially 5-nitrosalicylic
cornpounds forne<l

aci<l and a small amount of the 3-nitro isoner'

In a study of
TARLE
nolar ratio of nitrate:salicylic acid' no
2
absorbance versus
of the Salicylic Acid, Dissinilatory Nitrate Reduc-
further increase in absorbance was noted beyond a l:l nolar
Cornparison
tasl, and Phenoldisrrlfonic Acid llethods for Nitrate Analysis
of Extracts from Dried Plant Tissues'
ratio (Fig. 3)' This also suggests that nitration occurs
prinarily at one site on the salicylic acid rnolecttle'
Dissimilatory Phenoldisulfonic Salicylic
Sanp I e Nitrate Reductase Acid Assay Acid Assay Noninterference of other ions in this nethod l{as
not in-
AssaY
vestigated' Perhaps the rate of reaction Hith nitrate ions
us No;-N/s dry wt
is rapid enough to preclude binding of other ions at
the re-
oat leaf blade 12,657 1 2,1004 l5,l78b 14 ,870 t 3704

t 2,700 18,230 15,873 t 620 active site.

Oat cultn 14,610
This method is currently heing used r'rith success for
Corn leaves 2,257 ! 43 2,O25 2,471 ! 59

! analysis of soil and water sanrples' The rnethod should he

Corn sten 16,150 ! 360 I 8,200 16,3?l 470

1,672 t easily adaptable to autonated systens of analysis'

Corn sten l,7lS ! 106 I ,402 67

tlean and for three replications'

standard deviations
bA
single detenninetion was nade with this lethod'

78
77
 DETERMINATION OF NITMTE IN PI.ANT TISSI'B
CATALDO ET AL.

3. Beevers,L.: Schrader, 1,,E.; Flesher, D.; llageman, R.l!.

o.7 Plant Physiol. 40:691-695 (1965).
E
c o.6 4. Bremner, J.[tl. In "Methods of Soil Analysis", Part 2,
o Black, C,A., lid., Agronony, No. 9, Aner. Soc. of Agron.,
e o.5 l'tadison, l,I , 1965, pp, l2l6-1219.
5- Carlson, R.lrl.; Keeney, D.R. In "Instrlunental i\lethods for
t, o.4
e,
c Analysis of Soils and Plant Tissuesr', Walsh, L.lt., Ed,,
o
TI o.3
Soil Sci. Soc. of Arner., Madison, lttl , 1971, pp. 55-57.
o
6. Carvse, P.A., Analyst 92:3ll-315 (I967).
oct o.2
o.l 7. llolty, J.c. ; Potworowski, lt.S. ; Fnvir. Sci. Tech. 6:835-837
(1e72).
oI, I
o o.5 l.o 1.5 2,O 2.5 E. llcNamara,
J. Agr.
A,L.; lleeker, G.B.; Shaw, P.D.
Food Chen. 19':229-231 (1971).
llaf,enan, R.l!.
Molor Rotlo (NO;/ sA)
9. llorrison, R.T.; Boyd, R.l'l . "Organic Chenistry", 2nd ed,
Allyn and Bacon, Inc., Boston, llass,, 1966, p. 344.
FIG.3 10. Stalcup, ll.; Willians, R.l{. Anal, Chem.27:543-546 (1955).
Absorhance vefsus rrrolar ratio of NOl-N:salicylic acid. ll. Itlest, P.l{.; Ramachanrlran, T.P. Anal.' Chim. Acta 35:317-524
(1966).

REFER[NCES

l. nuu,. r, r tura r es earch Se rv i ce,

l":n"I::l
(r.). ::_,
r,epartment "--lilll cuand
of Agriculture,
R
the Agricrrltural ;;p;"-
rment Station, UniversitZ of lrisconsin_ltoai.on.
supported hy the College of Agricrrlturaf-ana n"r""i"i-
life Sci;;;;r,
University of Wisconsin-lfaaisin, trv'Uatn Coop".ative
nent l2-14-100-l0,ggg, and by un Agree_
SchooI Research Comnittee. I\lention the urtl Graduate
""".a-i.om
of a trademark or pro_
prietary product does not constitute
" tr"""na"u or warranty
of the product bv the U.S. Departn"nt-ol-igri"ulture
and
1::: inply iis approval
1^t that may also to the exclrrsion of other
products he suitable.
2. Forrner Postdoctoral Fellow, Graduate
Research Assistant,
Associatc professor
"t ne.ono,ni,-;;;
Departnent of Asriculture. ;i;;;;""h c.henist, rJ.s.
gli is atso-issociate professor
of Agronony).'present address or-onci'i"ttelIe, pacific
Northwest Laboratories, Ecosysterns-'n"p"ili"na,
l{ashington 99JS2. Richland,

79
80

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