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A NEW SPECTROPHOTOMETRIC METHOD FOR THE DETERMINATIONOF NITRITE IN'SEA WATER
Office of Naval ResearchContract N8onr-520/mProject NR 083 012January 1952
Technical Report No.8
, :.-
UNIVEE~ITY OF WASHINGTON OCEAN'OGRAPHIC LABORATORIE-SSeattle and Friday Harbor, Washington
Reference Na o 52-1
l!. NEW SPECTROPHOTOMETRIC METHOD FOR THE!?iTEmlINATION OF NITRITE IN SEA WATER
by
Kannsth Bendschneider and Rex J 0 Robinson
Technical Report No u 8
Office of Naval Resea?chContract NSOZlrea;20/II1
Project 1m OS,3 012
~TRODUCT!ON
TABLE OF CONTENT~
i
1
THE PROPOSED METHOD 2
Apparatus 2
ReagentsSulfanilamide 2NIlP(l-naphtlwl)ethylened:Lamine dibydrochloride 2
Standard Nitrite SolutionsStandard Solution I 2Standard Solution II :;Standard Solution III :3
Method ot .A!1alys1s 3
Optical Density Curves at V~ious Wave Lengths 4
Effect of the OrdaZ' of Adding Reagents upon theColor Development 4
Effect ot Hydrogen Ion Concentration upon ColorDevelopment 6
Effect of Reagent Concentration and Time Intervalbetween the Addition ot Reagents upon·ColorDevelopment . 6
Velocity ot Color Davelopment 9
Stability of the Reagents and the Color Producedby the Reaction· . 11
Effect of Temperature upon Color Intensity 11
Effect of Salinity upon Color Intensity 13
Calibration Curve 13
CONCLUSIONS
LITERATURE CITED
17
is
\
LIST OF F'IGURE§
$ NO" TITLE PAGE
1 0 Absorption Spectra ot the Diazo Compound 5. no Effect of Hydrogen Ion Concentration upon the
Maximum Color IntensiV 7
II;l:o Effect of Reagent Concentration and 'lime Intervalbetween the Addition ot Reagents upon ColorDevelopment g
A
IVo Velocity of the Color Development ·10
Vo Calibration curves 16
LIST OF TABLES
The Effect. at Temperature on the Maximum ColorIntensi't;y
The Effect of Salinity upon the Maximum ColorInt~n.cs1ty Produced by the Accepted Procedure
12
:
!,.
.A NEW SPECTSOPHOTOMETRIC METHOD FOR THEDETERMINATION OF :NITRITE IN SEA WATER
A spectrophotometric method employing sulfanilamide and
m"(l.naphtby~)etbylened1am:1nehas been proposed for the determination
of ni.trite in sea water. The effect or the order ot addition ot
reagents, aoidity~ reagent concentration, and temperature on the
speed of. colar formation and max'mum optical density has been dis
cussed0 It has been shown that saliJdty has no effect on the maxi
mum color intensity. The color develops rapid~ in either sea water
or fresh water and is stable for appr~tely two hourse Calibra-
tiOD curves have been prepared which show that the ooncentration ot
Ditrite is directly proport1oll8l to color intensity0
:
A NEW SPECTROPHOTOME~'RIC METHOD b'OR. THEDETERMINATION, OF NITRITE n~ SEA ~ATER
INTRODUCTI0!'i .
The most widely accepted method for the determination of
nitrite in sea water is based on the diazotization of- sulfanilic acid
by the nitrlte ~ with subsequent coupling to d. -naphthylam:i.ne to
give a rose-colored dye whose color intensity 18 proportional to the
nitrite concentration (1,5)" This method is rather slow, and spec-
trophotometrically the results are not as consistent as might be
desiredu
Shinn (6) used sulfanilamide as the diazotizillg agent and
N-(l-naphtbyl}ethylenediamine dihydrocbloride as the coupling agent
in the determination of nitrite in fresh water.. Equation (1)
represents the diazotization reaction!> The product of the coupling
reaction is not definitely known but equations (2) and (28) represent
possibilities:
(1) NH2S02C6H4NH2oHCl + HONO~ NH2S02C6H4N== NCl + 2H20
(2) :NH2S~C6H4N=Nel + C10~NHCH2CB2NH202HCl~
NH2S02C£,H4N.:::: 1-mtCH2CH2NH{C10~) t)2HCl + Hel
(26) NH2S~C6H4N=NCl + CloH7NHCH2CH~202HCl --)
NH2S02C6H4N =~O%NHCH2CH2NH2~2HCl + Hel
Shinn claimed more rapid color development a.nd greater
color stability with these reagents than ~1th th~ ~ustomary reagent~~
Kershaw anti Chamberlin (2) modified the Shinn procedure whioh resulted
in increased sensitivity and taster color developmento The present
paper reports the results ot a spectrophotometric study of this method
as applied to the determination ot Ditrlte in sea watero
THE PROPOSEJ) METHOD
Apparatus
A Beckman spectrophotometer, model DU, withCorex cells
ot 10 cm optical path, _s used ttn.' optical.density measurements in
this invest1gationo A slit width ot 0 0 10 mm was found to give satis
tactGr1' r~su1tso DiBtUled water was used in the reference cell
except asothendse notedo
Reagent.
SUlfAn",m'de
Five grams of sultanj]am1 de, NH2~C6H4~' are dissolved
in SOO ml ~ 1 0 2 N qdroch1oric acido The solution should be stored
in an amber, glass-stoppered bottleo
NCD(1:naphthvl)ethylened1am1ne dihVdroch1oridl
A halt gram of the compound, ClOB7NHCB2CB~2,,2JI}19 is
dissolved in 500 ml of' distilled watere The solution must· be stared
in an amber, glass-stoppered bottle·and kept out of direct sun]'ghtQ
Stalldard Ritrih Solutions
stapnArd Solution I
Pure sodium rdtrite 1s dried at 110° C and cooled in a
desiccator0 Ot the dried compound, 0 0 345 gram is dissolved in
distilled .ter and the solution diluted to one litero A few drops
4,
of chloroform are added to prevent bacterial growtho One ml oontaina
SeOO.P8 atoms of nitrite..Jlitrogeno
Standard Solution II
11va ml of Standard Solution I are dilu·ted to 500 ml with
distilled water o One ml contains 0.05}lg atom of nitrite-nitrogano
Standard Solution, III
Standard solutions or the desired concentration are prepared,
itlhen9Ver needed, by dUution of Standard Solution II with distilled
water over the range Oc.05 to 1 0 00 pg atoms of nitrite-Ditrogen per
liter of.' solution\) Nitrite-free sea 'water or sodinm chloride solution
may be used for dilution or standard solutions, but it is not neces
sar,y since the presence of these salts has little effect on the final
color intensityo
Standard ~olution I is usually stable for several weeks Cll
Standard Solution II is usually quite stable but variations in con"
cantration occasionally occur in a tew days and careful control is
necessaryQ Standard Solution III is not very stable and needs ~o be
prepared tresh whenever used o
Method of Anall~'Ji
One ml of the au1tanilam~.de reagent is mixed thoroughly with
50 ml or sea water sample followed by 1 m1 of N-(lewnaphtl\Vl)ethylene
diamine dihydrocbloride reagent within 2 to 6 minuteso The solution
reaches its maximum color intensity in about 10 minutes and remains
stable for approximatel1 2 hours o The optical dene1tr should be
measured during this period by means of the spectrophotometer, with
a wave length of 543 JD}llil Th~ nitrite concentration is determined
trom a calibretion cUrve previously prepared using standard nitrite
solutiolJ.s.
EXPERDE}I'fAL
Opti~.sl Den~itl Curves at Various frave Lengtha
Absorption by the diazo compound was measured at various
wave lengths or. each of three solutions containing 001, 1 0 0 and 2~O
pg 8t.omB or nitrJ:te-nitrogen per liter in 8 solution of S=-34G3%o o
.Maximum absorption occurred at 54.3 m}1 88 shown in Figure I o In all
subsequent experiments with these reagents optical density observaco
tiona ~ere msde at this wave lengtho
.§treet or the Order of Adding Reagents upqn the Color Deyelopmen'\;
Various orders ot adding the sulfanilamide, N-(l-naphthyl).
ethylenediamine d1bJdrochloride and hydrochlorio acid were investi
gated~ It wes determined that the maximum color development occurred
Whei.l the sulfanilamide and hydrochloric acid were mixed with the
sample before the coupling reagent was added o It is essential that
the diazot1zation reaction, represented by equation (l)g be como
pleted before the coupling reagent 1s added; otherwise, the un
diazotized nitrite reacts with the amino group of the reagent with
e resultent reduction in color intensit,., acoording to Rider and
Mellon (4) e A reagent containing all three reagents wsa tound to
be impractical apparently because of the reaction of nitrite with
the amino group of th~ ooupling reegento
1\
1.200.....--------------...
>- 0.800I--enzl1JCl
--I• «
u-I- 0.400a.0
0.000
400 500 600
WAVE~LENGTH (mJJ)
:rmtJRB I
Absorption Spectra of the Diazo CompoUDdo
Curve A. 2 0 0 JII atoms of B02-B ptesen't per liter of solution.Curve B. 1.0 JIg atom of -02--11 present per 11tar of solution.Curve C. 0 0 1)Jg atom of IilOi-B present per liter of solution.
-5-
Effect ot HYdrogen Ion Concentration upon Color Development
A series of 10 0 per cent sulfanilamide solutions was preo
pared with h7droch1o~ic acid concentrations ranging from 0 0 0:3 N to
60 0 No One ml ot 8ach ot these solutions was added to a 50 ml stand-
ard Jdtrite sample, 100 pg atom per litero The sample was well
mixed and 1 ml ot a 00 2 per cent solution of N-(l-naphthyl)ethylene-\
d1am1ne d~drochloride reagent introduced 60 seconds atter the
addition ot the sulfan:1lami de reagento '!'he maximum color density
and the pH of each sample were determinedo A plot of the results~
Figure II, indicates that with 10 0 per cent su11'ani1 em'de reagent,
and lmiDute d1azotization time, a pH of 2 0 0 or less is required to
produce maximum color intensityo
~ct at Reagent Congentration and Time Interval between the Additionot Reagents upon Colar Development
The time required tor complete diazotizat1on was shown to
var'7 with the sulfanilamide concentration when the pH is held constanto
nn,.ee solutions were prepared with OoS, 100, and 2 0 0 per cent sulta
Dilamide and 80 ml of concentrated qdrochloric acid per liter ot
solution0 One ml of each of these solutions was added to a correo
sponding 50 ml standard nitrite sample, 1 0 0 pg atom per liter" The
sample was well shaken and 1 ml of 001 per cent N-(l-napbtql)ethyleneca
dfamine d1h7drochloride introduced .30 seconds atter the addition of
the first reagento This procedure was repeated several times :in the
same marmer except that the time interval between the addition ot the
reagents was variedo A plot, Figure III, ot the maximum color den..
siV of each sample against the time interval indicates the time
\.
>- 0.520r--enzwo--I<{
~ 0.480r-.0..o
0.440o
lit
2.00HYDROGEN ION (pH)
"PIGUBB II
Iffect of !JJdrogen Ion Concentration upon the Max1mum Color IntensiV.
1.0)11 atom of ~--. present per l1ter of solution.
..
4.00
1·
I.'III
0.490
>I--enzwo
ci. 0 .470(.)-I-a..o
O.450--....01--...-.--......------6~O~--..---~~- ......-~~
TI ME (SECON OS)
FIGURE III
Btteot of Beagent Ccmcentration aDd Time Interval between theA4CU.tloD. of Reagents upon Color Developnellt.
CurVe' A." 2.0 per~c:ent 8U1tan1'lAm14e~.Curve B. 1.0 per cent 8t11tanf]eYdde. .Curve c. 0.5 per cent sultan'lamide.1.0 pg atom of moa--Il present per liter of Bolutiono
•
,-
required for complete diazotization with different concentrations of
sulfsn1]amide 0 All optical densities below the highest value reveal
incomplete diazotizat1ono
The coupling reaction 1s bimolecular and consequently the
reac~on velocity is dependent upon the concentration ot either
reacting substance 0 Boever, by using a ,rather large excess of
N-(1-naphtb11)eth;ylenedi am1 ne dihydrochloride the reaction· velocity
can be made practically independent or the concentration ot this
reagent, and the r.eaction becomes a pseudo-tin1moleeular one o One
ml or a 0 0 04 per cent solution ot the coupling reagent, which reprsE9nts a
twenvwtold excess over the diazotized sample, was determined to besufficient to transform the reaction into a psaudo-unimolecular one o
Essentially. the same reaction veloe!ty .8 observed in the coupling
reaction wheu using ODe ml of either a 0 0 04 per cent or 00 4 Par cent
solution or .N-(l-l18ph~l)etbylened1amine dihydrochloride Q
!.'U:..oc1tx of eolor Develqpment
The veloc1t,' of color development is greater with the prOc:»
posed mothod than with the method currently in use I) Full color
development is attained in about 10 minutes with the proposed reagents
in sea water medium whereas about 30 minutes are required with sulfa
nilic acid and qoDaphthylam'neo With the latter reagents about 90
minutes are required tor full color development in tresh water medium
whereas with the pt-opoeed reagents there 1s no difference in the
veloc1V ot color development 1D either fresh or salt .tar mediDo
TJrpical kiDeUc curves are shown 1D Figure IV0
,bI
'..
>-..... 0.400enzlLIo.-J~(.)
i= 0.200C-o
o 0
•
30
. --
90TIM E (M IN UTE S)
FIGUBE IV
VelooiV of the Color DevelopaeDt.
'.
150
Curw A. Proposed reagents in sea water medium.Curve B. Aocepted reagents in sea water meMUIl.Curve C. Accepted reagents 111 tresh water 11ied'\1IDo1.0~ atom moa---m pz-eseDt per liter of 801uUoll.
Stability of the Reagents and the Color Produced by thL,Reaction
The acid-sulfanilamide reagent as used in the determination
is very stable!J no decomposition being evident in a period of three
mon:Ghs 0 The N-(l<Qnaphthyl)ethylenedisndne dihydrochloride reagent
slow~ decomposes but may be stored in an amber glass bottle for at
least two weeks without appreciable d1scolorationo Direct sunlight
g~eatly accelerates the formation oof the brown decomposition producto
Under conditiona o£ artiI."'icial light or bright daylightI
(but p"'otected from direct sunlight) the color produced by the reaction
is stable for about two hours, then slowly fades v In direct sunlight
the color changes rapidly to a redder hue with an increase in optical
density; t~t is; the maximum absorption wa~le length ehif"f;s from
543 JD}l to about 5.30 Inp,o
The diazotized sulfanilamide is also somewhat unstable and
the time interval between the addition of reagents should not be pro°
longed (I With a l5""'minute time interval no decrease in maximum optical
density oceurs~ but when the time interval is incrnased to 30 minutes
a 5 par cent decrease in maximum optical density -results u
Effect of Temperatm-e urJOn Color Intensipy
The color intensity is also dependent upon the temperature
of the aolution~ In Table I are the results obtained when ni"urite
solutOions 1t containing 10 0 pg atom per liter,u were heated to different
selected ~emperaturesp treated with reagents and maintained at these
desired temperatureso After determjnjng the optical densities the
solutions were slowly cooled .to room temperature and the optical 0
t ,..
.!h
f,he Ef'gect otTemperature on theJJ1ax~um Color Intensit;y:
Temperature ot Optical Density at Final FinalCouplins Re8ctiRn ~§aotion Temperature Tem~ratPzt:! Optical Depsitx
C C
13 0 0 O~510 29 GO 0 0 505
300 0 0498 30 0 0- 0498
;10eO 0499 140 0 1>510
38.&8 0489 29.0 0498360 0* 9491
~ 42.<1:3 ~492 2905 0;01a
49.08 .~483 29iJ5 eSCS
68:0 0 ,0474 29,uO .491
70.0 0 oM4 29 00 0480
'* Sample warmed from 29;10°0 to 36~OoC and the density redetermined tJ
1 0 0 )lg atom o~ ni·tri·~e..nitrogen present per liter or solutiono
II
c1ensitj.es redetermined", The oolor intensities varied inversely '\~:t.th
temperatur:'3 f.' When the solutions were again brought to the s£m~
temperatura, the color intensities became essentially the same except
for those which had. been heat9d above 50° C when some decomFcsition
apparent~ had occurred~
~e£t of Salinity UPOrl Color IAtepsi1;z
iatrite solutions were prepared in distilled water medium
and in various synthetic sea wetar media, prepared aocording to Lyman
and Fleming (3) 0> Identical optical densities were obtained for m'hr-ita
solutions of equal eoncentrat~.on in either med.1.um"
Liko'tiise aalinity was found to have no effec·t; with the
reagents now currently used if the ~-naphthylamine was added after
the sulfanilic acid" However~ it these reagents were added as a
mixture ~ a slightly lesser optical dens!ty was noted a t the lower
salinities~ Table IT.o With the range of salinities normally en....
countered in marine work~ this e.t:f'ect is practically insignificant
except in very critical worko
Calibr!tion Cy;,m
Figure V shows calibration curves with both the pro-
posed and accepted res~ntso Since Curve A is linear, the color
produced is directly proportional to the nitrite concentration in
conformity with Beerlls lawo Within the llmits noted abov8 p the slope
of the curve is constant under ditterent conditiona ot pHs reagent
concentrat1on~ time interval between reagents1} and standard solutions
prepared with distilled water or DitriteEDfree see water"
~.BLE II
The Effect of Sal11'lity upon the Maximum ColorIntensity [-:roduced gy the Accept~d Procedure
Sample Optical Density Opti~~G.l DeneitySalinity with Mixed wi·th Separate__.oLQQ _ I Heo.gents .-.Jleage:n~f!....._
34..3 O~208 0 0 210
24:;0 ,204 .,209
17 0 2 (1204 ~209
10 0 6 0201 0209
:305 .,199 ...209
~ 01,6 0193 0)208
00 0 0192 ;,209
Density Observations at 520 Jl1).l.0 Samples contained0050 FE atQm of nitrite-nitrogen per liter of solution o
to
,
As iridicated in Figure Vs some 8 bsorpt:i:jn occurs at zero
nitrite concantration u This probably is due to nitrite imptn."ity in
the reagents or in the dist111ed water since neither reagent causes
absorption at wave length 543 ~ when added alone to distilled watero
Redistillation of the Viatar ~ made alkaline with sodium hydroride 9
usually reduced the observed optical density Q Distilled watf'~ plus
reagents may be used in the reference cell to compensate· tor the
blank, but this is not recommended since it introduces the possi
bility of variations in the absorption characteristics of the refer
ence cell due to decomposition or contaminationo
In Figure V the greater slope of Curve A in comparison with
Curv·e B indicates that the proposed reagents are more sensitive than
those now used o Thus with the proposed reagents~ smaller amounts ot
nitrite may be estimated and smaller differences in nitrite concen...
tration may be detected o
It 'Was also noted that somewhat greater consistency was
obtained with the proposed reagents o Definite variations in the
slopes or calibration curves were obtained occasional~ ~1th the
mixed sulfanilic and d.~naphthylam1ne reagents o
o
..-. • {aJ4. .. •
0.600>-I--enzwo...Jc:{
D 0t i=
Q.00.200
o 0.20 0.60 1.00 1.40NITRITE-NITROGEN (~G. ATOMS PER LITER)
FIQ11BE V
Ce)4bration CurveS o
Cam A. Proposed method B t 543 muGCarve B. Aaaapte4 method at 520 BlU.
..
CONCLUSIONS
By the proposed method full color is developed much more
rapi<1ly than by the method now in use 0 This color is amply stable
fOl~ convenisr.t measm-ement of optical density, but is not as stable
aa the color by -the method now in use 0 The proposed method is more
sensitive than the accepted methodo With t;he proposed reagents the
color intensity is proportional to n1trite concentratioD over the range
of nitrite ooncentrations norma~ expected in sea watero
a
i
1.. ILOSVAY, Lc18S9u Determination or nitrite in saliva and exhaled air.:.
BUlle SOCo Cbimo Fral'lca~ (3) g:; 388.w391o
2.:- KEltSHAW, ~l() F~ and N& So CllAMBEHLIN1942" Determinatlon of n1trites Cl Industr Q Engng., Chern .. ,
Analt. JUl", ,l4g 312-3130
.3;, LYN'!AN sa J 0 and R" H0 FLEMING19400 Composition of sea water~ J~ ~ar~ Research j ~:
134"'146"
4e HIDER, Bu .F'., and M0 Go MELLON1946" Colorimetric determS.nation of nitritesD Induetr Q
Engngc Chem01 Anal~ Edo 9 ~~ 96~99o
50 ROBINSON, Ro Jo and To G~ THOMPSON19480 The determination of nitrites in sea water~ .
j'G Mar-v Research~ 2g 42e.4e o
6e SHINN:1 Me 8 0
19L,,10 it colorimetric method for the determination of nitri'iie u
Industr .. Engngo Cham,., Analc. Edo, U: 33=35.:;
NcoQ.QJ£~~.
NO e
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DirectorChesapeake Bay Inst:l·tutoBox 426A, RFDIl211.nnapolis ~ Marylatld
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Head, Department of Oceenography'Univ'3rs:i.ty of WashingtonSea t tle ~ Wa shingiion
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Texas A" & lIdQ CollegeCollege Station, Texe.3
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Brown UniversityProvidence ~ Rhode Islmld
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