INTRODUCTION

1 Research Paper 5. Cooperative Fisheries Re­search Staff of the Territorial Board of Agricultureand Forestry and the University of Hawaii.

2 Department of Chemistry, Un.ivers.ity of Ha­waii. Dr. Dean is now at the University of C!re­gon; Mr. Hawley is a student at George W~ShlOg­

ton University Medical School. Manuscnpt re­ceived December 2, 1946.

FISHPONDS IN THE HAWAIIAN ISLANDS areshallow brackish ponds which are frequentlyfed by underwater springs as well as surfaceflows of fresh water and sea water. Thechloride content of .the water is an indicationof the quantity of sea water present and isone factor which affects the growth and sur­vival of many of the plants and animalsutilized as food by the fish. The presence ofdissolved oxygen is absolutely essential foranimal life in the ponds. Low oxygen con­centrations in some regions indicate stagnantwaters and bottom putrefaction which makethese areas' unfit for fish. In the course offisheries studies in the Hawaiian Islands itwas therefore necessary to make a large .number of salinity and oxygen determina- .tions on fishpond waters.

Since both chloride and oxygen may varyseveralfold in a single pond,and since mostliving organisms are insensitive to concentra­tion changes which are less than 1 per centof the normal value, it is not necessaryto make extremely accurate determinations..Methods were desired ·for the rapid analysisof chloride and oxygen at the side of thepond so that any unusual observations couldbe checked before one left the pond.

The apparatus described in this paper wasdesigned to provide sufficient equipment andreagents for 25 chloride, and 50 dissolved

A Chloride and Oxygen Analysis Kit for Pond Waters l

R. a DEAN and R. L. HAWLEy2

oxygen determinations. Two auxiliary.half­liter bottles contain enough reagents foran additional 50 chloride determinations.Chloride up to 25 gm. per liter (sea wateris about 20 gm. per liter) can be determinedwith an accuracy of 0.06 gm. of CI per liter.Dissolved oxygen up to 20 cc. per .liter canbe determined with an accuracy of 0.1 cc.per liter on a sample of 10 cc. of water. Theapparatus weighs ab?ut 25 lb. and ca~ beused in a rowboat, If necessary. A singlechloride determination requires about 3minutes; an oxygen determination requiresabout 8 minutes.

Although there are several well-knowntitrimetric methods for chlorides, none ofthem is suitable for use on micro-samplesout-of-doors where it is difficult to observea colorimetric end point. An electrometricmethod of detecting the end point whichsubstituted a galvanometer needle for thecolor change appeared to be more suitablefor our purposes. None of the publishedelectrometric methods was quite satisfactoryand an entirely new method was developedwhich will be dealt with in another publi­cation (Dean and Hawley: ' unpublished).This method makes use of two wire elec­trodes, a galvanometer, a few radio re­sistances, and a dry cell. A relatively lowresistance circuit is formed which is notsensitive to high humidities, although itshould not be soaked in water.

Krogh (1935: 131':"133) has described. amodification of the Winkler method for dIS­solved oxygen which was well suited to ourpurpose, with slight modifications. Kroghused the conventional starch indicator to de­tect the end point. . We have substituted

108

Chloride and Oxygen Analysis Kit - DEAN and HAWLEY 109

Foulke's dead-stop electrometric end point(Foulke and Bawden, 1926: 2045 ff.), be­cause the starch iodine indicator is unreliableabove 25° C. and our field temperatures fre­quently exceed 30° C. Essentially the sameelectrical circuit is used for both the oxygenand the chloride end points: only the elec-trodes are different. '

The unknown solutions are titrated withstandard solutions from a micrometer syringeburette (see Trevor, 1925: 1111; Dean andFetcher, 1942: 237; and Dean: unpub­lished). This burette has many advantagesover conventional gravity-feed burettes. It ismore compact, there is no drop error and noparallax error, and better than 0.1 per centaccuracy is possible on a total volume ofonly 1 cc. of reagent. Micro methods areobviously necessary to conserve reagents if asmall apparatus is to carry enough to permit50 or more determinations. Two syringesare used interchangeably with the samemicrometer head in this apparatus, so that itis not necessary to clean and refill the burettewhen changing from chlorine to oxygenanalyses.

DESCRIPTION OF THE APPARATUS

All of the equipment is 'contained in awooden box which measures 9 X 9 X 12inches ' (see Fig. 1). The electrical wiring isenclosed in a mahogany case which is firmlyattached to the right-hand side of the box.The galvanometer is a needle type of instru­ment which has a sensitivity of 0.25 micro­amperes per ' scale division and a criticaldamping resistance of 1,800 ohms.

The electrical circuit is shown schemati­cally in Figure 2. The two switches Sl and S2are combined in a telephone type of toggleswitch. In the central position both switchesare open and no current flows. When theswitch is moved to either side, current flowsfrom the battery through the series of re­sistors in the upper line. Suitable taps takeoff about 10,200 and 1,018 mv. to the Pt,

Ag, and Standard Cell terminals respec­tively. Variations in the internal resistanceof the dry cell can be compensated for byadjusting resistor R2 when the switch isthrown to the left. In this position the volt­age of the standard cell opposes a fractionof the voltage from the battery. Resistor Bis adjusted until no current flows throughthe galvanometer. In actual practice it hasbeen found that the voltage from a flashlightdry cell does not change significantly, evenwhen the circuit is left open for 2 days. Itwould have been satisfactory to omit thestandard cell and variable resistor B entirely.The potential at the chloride electrodes couldbe checked before each series of measure­ments, as is described below. .

The electrodes are constructed as shownin the insert of Figure 2. Short pieces ofsilver or platinum wire are attached to stiffbronze wire with hard solder. The wire elec­trode is then sealed in a straight glass tube.It is not difficult to seal platinum into softglass. Silver wire of 22 gage can also besealed into soft glass but some of the -sealsbreak soon after sealing. After the glass issealed onto the electrode wire, both the glasstubing and the bronze wire can be bent inthe ordinary manner, since bronze wiresoftens at red heat.

The stiff bronze wire is passed through a' hole drilled in a small radio jack plug asindicated in the diagram. The four electrodeleads-Ag, Cu, and two Pt-are brought tojacks on a mounting plate at the left end ofthe box under the end of the burette. Wheneither pair of electrodes is plugged into itscorresponding jacks, the electrode wires justreach the center of the titration vessel. TheAg and Cu jacks are further differentiatedby color to reduce the danger of inserting anelectrode in the wrong terminal.

The micrometer-syringe burette consistsof a 1.5 cc. glass hypodermic syringe fittedin a frame to which is attached a l-inchmachinist's .micrometer graduated in 1,000parts. The burette assembly is described fur-

110 PACIFIC SCIENCE, Vol. 1, April, 1947

FIG. 1. Analysis kit for chloride and oxygen. Chloride reagents and syringes to the right; oxygenreagents and syringe to the left. E, electrode holder. The chloride electrodes are just above the slide·rule . P, micrometer burette with syringe to hold sodium thiosulfate reagent. The silver nitrate syringeis above chloride electrodes. N , galvanometer. Z, toggle .switch. A, standard cell. T, rubber bulb inbottle of distilled water for washing. The syringes and slide rule fit in clips inside the lid of the box.Most of the equipme nt remains in the box during the analysis.

Chloride and Oxygen Analysis Kit - DEAN and HAWLEY 111

ther in Dean and Fetcher (1942: 237; seealso Dean: unpublished). For the oxygendetermination a solution of thiosulfate isused in the burette and a 2-inch 22-gagehypodermic needle bent downward at 90°serves as the delivery tip. The chloride de­termination requires a strong solution ofsilver nitrate, which corrodes all availablemetals, so that a glass tip must be used. Inthis apparatus we used a bent glass tubeattached to the burette by a number 0 one­hole rubber stopper."

3 The Ace Glass Company, Vineland, N. J., isnow able to supply ground glass tips to orderwhich will fit on a standard hypodermic syringe.

The titration vessel is a shortened test tubeabout 25 mm. in diameter and 70 mm. long.It fits in a wire test tube holder which isattached to the electrode jack assembly, Theburette tip and the two electrodes dip intothe solution in the titration vessel. In addi­tion there is a glass tube to introduce airbubbles for stirring. Air is forced by anaspirator bulb into a t-liter can whichserves as an air reservoir, and flows fromtherethrough a thin rubber tube to the glassnozzle of the air stirrer. The aspirator bulbcan be conveniently squeezed by the lefthand of the operator during a titration.

A reagent shelf over the air reservoir

DRY

CELL500

10,0001-------

DETAIL OFELECTRODES

Pi orAgwire

~=~Bronzewire inglass

tadiOjack

MICRO AMPS

FIG. 2. Wiring diagram. S, and S. are two parts of a double-pole, double-throw telephone-type:toggle switch. Wires run from the Pt, Ag, and Cu terminals to the electrode holder.

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holds five 60-cc. screw top bottles and two10-cc. screw top vials. In addition, there areseveral larger reagent bottles in the bottomof the box and a bottle of distilled water forwashing electrodes and glassware. A glasstumbler is fitted under the electrodes for awaste jar. A rubber ear syringe is fitted witha glass delivery tip bent at an angle of 1350 •

This syringe can be filled from the distilled.water bottle and the water in it then directedin a stream into the inverted titration vesselor onto the electrodes to clean them.

The reagents are all contained in 'screwcap bottles or vials. The screw caps are linedWIth Polythene, which is resistant to all thereagents used in this work. Two of the vialsare fitted with rubber disks from 10-ml.penicillin bottles . The disks are held on byscrew caps which have been drilled withJ4-inch holes. The rubber disks on the .vialscan ~e repeatedly perforated by 'a hypo­dermic needle and the reagents withdrawnthrough the needle .

All the reagents 'and equipment used forthe chloride analysis are marked with blackpaint and the various reagents are identifiedby w.hite letters on the top, as well as by con­ventional labels on the sides of the bottles.The reagents and apparatus for the oxygendetermination are likewise marked with redpaint and white letters. The metal parts ofthe syringe pipettes and the burette are ofb~ass which was first dulled by a dip in silverfiltrate ·and nitric acid, and then coated withGlyptal varnish which was baked on. Thistre~tment produces a non-glaring, corrosion­resistant surface. The syringes are attachedby clips to the inside of the top of the box.A small box of filter paper squares, about 3cm. on a. side, which are used to wipe theburette tips and the electrodes, is also at­tached inside the top of the box. A rubberstopper is screwed inside the top at the right­hand side in such a way that the lid will notclose until the switch has been turned off.This arrangement insures that the batterywill not be left on when the box is closed.

PACIFIC SCIENCE, Vol. I, April, 1947

CHLORIDE ANALYSES

The chloride analysis depends upon thefact that the potential of a silver electrodechanges rapidly when all the chloride ionshave been precipitated by silver ions. Sinceit is impossible to measure a single electrodepotential, another electrode which is not

. sensitive to chloride ions must be present tocomplete the circuit. We have used a copperelectrode in the presence of nearly saturatedcopper sulfate. The potential between thesetwo electrodes is about 200 mv. at the endpoint. The potentiometer circuit supplies200 mv. between the Cu and Ag electrodesand, at the end point, no current will flowthrough the galvanometer.. Before the end point is reached the poten­

tial between the electrodes will be less than200 mv. and the galvanometer will be de­~ected to the right. There is a large protec­tive resistance in series with the chlorideelectrodes so that the galvanometer may be~eft continuously in circuit. As the end pointIS approached the galvanometer needle ap­proaches zero and is deflected to the left assoon as the end point has been exceeded.The deflection produced by the addition ofone or two burette divisions of silver nitrateis greatest at the end point. This fact can beused to set the potentiometer to the cor­rect. potential. The potentiometer is adjusteduntil the galvanometer indicates zero. Thegalvanometer deflection is then noted afterthe addition of two burette units of silvernitrate. This procedure is repeated until thegalvanometer deflection is a maximum. Thepotentiometer is left at this setting for sub­sequent titrations.

The. silver .nitrate reagent contains 4 percent silver filtrate and 0.5 per cent nitricacid. The burette is filled by bringing thereagent bottle of silver nitrate, marked witha T on a black screw cap, up under theb~rette tip. The plunger of the syringe isWIthdrawn by unscrewing the micrometerand silver nitrate is sucked into the syringe:

Chloride and Oxygen Analysis Kit - DEAN and HAWLEY 113

Any air bubbles are displaced byrotating theburette in its clamp until the tip is upwardsand then forcing a little of the silver nitrate

.reagent out of the tip. The burette is filledup to the 1,000 mark on the micrometerand the tip rinsed with water.

Ten cc. of saturated copper sulfate isplaced in the titration vessel; a syringe withtip broken off is used to make the transfer.Commercial bluestone can be used for thissolution, although it may introduce an un­desirably high blank. Reagent grade coppersulfate is perfectly satisfactory. The coppersulfate solution is placed under the elec­trodes and the air stirrer is started. A smallresidual concentration of chloride ions mayproduce a galvanometer deflection to theright. If it does, silver nitrate is added untilthe galvanometer reads zero. The burettereading is recorded. The chloride sample,about 0.4 cc., is then introduced from aprecision syringe pipette (Krogh, 1935:130; Dean: unpublished). Silver nitrate isadded until the galvanometer again indicateszero and the second burette reading isrecorded.

Neither the exact volume relations of thesyringes nor the concentration of the silvernitrate need be known exactly. All these areevaluated as a single factor by titrating thesame volume of a known standard solutionof sodium chloride which contains exactly20 gm. of chloride ions per liter.

When the galvanometer has been broughtto zero a second time and the burette read­ing has been recorded, the air stirrer is re­moved and wiped. The burette is tipped upand the 'tip is wiped and the burette is re­filled. The electrodes and the burette tip arerinsed and the apparatus is ready for thenext determination.

The errors are of the order of 1 burettedivision or 1 part in SOO. Greater precisioncould be obtained by precipitating most ofthe chloride in a larger sample with silvernitrate from another pipette. A more dilute

solution of sil~er nitrate could then be usedin the burette. It might be advisable to carryout such determinations in more dilute solu­tions to avoid too much clumping of thesilver chloride precipitate. The accuracy islimited by the reproducibility of the syringepipettes. Krogh (1935: 130) reports anaccuracy of 1 part in 10,000 and we haveseveral syringes with an accuracyof 2 partsin 10,000.

OXYGEN ANALYSES

Oxygen is determined by its reaction witha solution of manganous hydroxide to formmanganic oxides. When all the oxygen hasbeen .absorbed, the solution is made acidand the manganic ions react with iodide ionsto liberate free iodine. The iodine is titratedwith sodium thiosulfate in the presence oftwo bright platinum electrodes. A potentialof 10 mv. is applied between these electrodesand a current will flow as long as there isiodine in the solution to remove electronsfrom the negative electrode. As soon as allthe iodine has been removed the currentceases to flow, except for a very small resi­dual current. As the end point is approached,the galvanometer deflection decreases fromthe left and the end point is taken when thegalvanometer indicates one unit deflection tothe left.

A io-«. syringe pipette (Krogh, 1935:132; Dean: unpublished) is fitted with a2-inch IS-gage stainless steel needle. Analuminum cam cemented to the plunger withVarno cement will engage a stop on theside of the syringe holder when the syringeholds 10 cc. An additional 0.2 cc. can beintroduced by rotating the cam away fromthe side stop and pulling back until theplunger reaches an end stop.

The syringe is first rinsed with Solution 1.This solution is made up by dissolving 90gm. of NaI and 40 gm. of NaOH in 55 cc.of water (Pomeroy and Kirschman, 1945:716) and all air bubbles are expelled. This

114

reagent is kept in a small rubber-capped vialmarked on each side with one white dot.The needle of the syringe is inserted intothe vial and the vial is inverted while thesolution is drawn in and the air bubbles areexpelled. This leaves about 0.1 ml. of solu­tion in the dead space of the syringe. Thewater sample is then drawn into the syringeuntil the cam on the plunger reaches the sidestop. The needle is then inserted into asecond vial, marked with two white dots,which contains Solution II. This solution ismade from 40 gm. of MnCl 2 , 10 cc. of6N HCl, and enough water to make 100 cc..This solution is drawn into the syringe bymoving the plunger from the side to the endstop.

The manganous chloride reacts inside thesyringe with the sodium hydroxide whichwas left in the dead space,. to produce a lightfluffy precipitate of manganous hydroxide.This precipitate absorbs oxygen, and in 4minutes substantially all of the oxygen willhave been absorbed. The contents of thesyringe are then discharged below the sur­face of 1 cc. of 6N HCl in the titrationvessel. The syringe is rinsed first with theacid solution to dissolve any manganoushydroxide remaining in the syringe, and thenwith two small portions of water from aspare titration vessel. The 6N HCl is con­tained in a 60-cc. bottle fitted with a rubbermedicine dropper. One dropper full is about1 cc.

The titration vessel now contains iodineequivalent to the oxygen which was presentin the water. The iodine is titrated with asodium thiosulfate solution. This solutionmust be made up fresh every week by dilut­ing to 60 cc. one medicine dropper full ofa 60 per cent solution of sodium thiosulfatecontaining 1 per cent borax. This concen­trated solution is quite stable. Most of theiodine is discharged with thiosulfate fromthe burette before the air stirrer is started,

PACIFIC SCIENCE, Vol. 1, April, 1947

because the air might otherwise remove someof the iodine.

The thiosulfate solution and the volume­tric apparatus are standardized by the use ofa standard solution of KIOs. This solutionis 0.0004167 molar and is equivalent to14 cc. (STP) of oxygen per liter. The deadspace of the syringe is first filled with Solu­tion I. The syringe is then rinsed with acidcontained in the titration vessel and filledwith the standard iodate solution. The iodatereacts with the iodide and acid to liberateiodine in the syringe. The iodine is titratedwith thiosulfate, and the burette differencewhich is found corresponds to 14 cc. ofoxygen per liter. Blank runs on water whichhad been freed of oxygen by hydrogen andplatinized asbestos showed that the endpoint and .other errors are less than 0.1 cc.of oxygen per liter.

The oxygen concentration is calculated inessentially the same manner as that used forthe chloride. The initial burette reading isalways taken as 1,000. This procedure in­troduces a small constant error of the orderof magnitude of 0.1 cc. of dissolved oxygenper liter .

SUMMARY

A portable apparatus which is equippedfor the determination of chlorides and dis­solved oxygen in pond waters is described.The chloride is determined by a new elec­trometric method. Oxygen is determined bya modified Winkler method and the iodi­metric end point is detected electrometrically.The volumetric apparatus consists of preci­sion syringe pipettes and a micrometerburette. Up to 25 gm. of CI per liter can bedetermined with an accuracy of 0.06 gm.per liter. Up to 20 cc. of dissolved oxygenper liter .can be determined with an accuracyof 0.1 cc. per liter. Greater precision over asmaller range of chloride concentration ispossible, since the syringe pipettes can attainan absolute accuracy of one part in 10,000.

Chloride and Oxygen Analysis Kit - DEAN and HAWLEY

REFERENCES

115

DEAN, R. B. Improved syringe pipet and microm­eter burette (unpublished).

---, and .E. S. FETCHER. Micrometer burette.Science 96: 237-238, 1942.

---, and R. 1. HAWLEY. A new rapid electro ­metric method for the analyses of chlorides (un­published) .

FOULKE, C. W., and A. T. BAWDEN. A new typeof end-point in electrometric titration and itsapplication to iodimetry. Amer, Cbem, Soc.Jour. 48: 2045-2051, 1926.

KROGH, AUGUST. Syringe pipets. Indus. and Engin.Cbem., Analyt. Ed. 7: 130-131, 1935.

--- Precise determination of oxygen in waterby syringe pipets. Indus. . and Engin. Cbem. ,Analyt, Ed. 7: 131-133, 1935.

POMEROY, RICHARD, and H. D. KIRSCHMAN. De­termination of dissolved oxygen, proposed modi­fication of the Winkler method. Indus. andEngin. Cbem., Analyt, Ed. 17: 715-716, 1945.

TREVOR; J. W. The micrometer syringe. Biocbem,Jour. 19: 1111-1114,1925.