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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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Depar-t;merrl'i or Eng::Lni:JoringuniV0~sity of Californi~

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l·h~ Oceanographic Inot:l:tu,teIi'lorida State Univers:1:t.:rTallahassee" Flol'ida

Fe S t. l!'ish & W:tldl:tf'.s ServiceF-oO ll Box .3830Honolulu, '.r.vfL~

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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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DirectorHaw::-i.ii Marine LaboratoryUniversity of HawaiiHonolulu, TI$Ho

Directo,;,l\1al~:tne La.boratoryUniversity of MiamiGoral ('~:lblaB~ Florida

Head, Department of Oa~)anogr2.:ph;f

Texas A" & lIdQ CollegeCollege Station, Texe.3

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Brown UniversityProvidence ~ Rhode Islmld

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