A Syringe Gas-stripping Procedure for Gas-ChromqtographicDetefmination of Dissolverl Inorganic and Organic Carbon in

Fresh Water and Carbonates in Sediments

Mlcnnnr, P. Sr^uNroN

Fisheries Research Board of CanadaFreshwater Institute, Winnipeg, Man. R3T 2N6

SrarNrow, M. P. 1973. A syringe gas-stripping procedure for gas-chromatographic deter-mination of dissolved inorganic and organic carbon in fresh water and carbonatesin sediments. J. Fish. Res. Board Can. 30: 1441'-1445.

A simple, rapid method for determining dissolved inorganic carbon in water is described.A 20-cm3 sample of water is drawn into a 50-cm3 polypropylene syringe and acidified by in-jection of 1 cm3 of dilute sulphuric acid. Twenty-nine cubic centimeters of helium at atmos-pheric pressure is injected into the syringe followed by 10 sec of manual agitation to partitionCOz between gas and liquid phase. The gas phase containine 6070 of COz from the sampleis then analyzed by gas chromatography. This method has been used to determine dissolvedinorganic and organic carbon in Canadian Shield waters and to determine total carbonatesin sediments.

SrarNroN, M. P. 1973. A syringe gas-stripping procedure for gas-chromatographic deter-mination of dissolved inorganic and organic carbon in fresh water and carbonatesin sediments. J. Fish. Res. Board Can. 30: 1441'-1445'

L'auteur d6crit une m6thode simple et rapide de dosage du carbone inorganique dissousclans I'eau. Un dchantillon de 20 cc d'eau est pr61ev6 dans une seringue de polypropyldne de50 cc et acidifi6 par injection de 1 cc d'acide sulfurique dilu6. On injecte ensuite dans la seringue29 cc d'h61ium d la pression atmosph6rique et on agite manuellement pendant 10 sec, jusqu'aupartage du COz entreTa phase gazeuse et la phase liquide. La phase gazeuse, contenant 6A/o{u CO2 de 1'6chanti1lon, est ensuite analys6e par chromatographie en phase gazeuse. Cettem6thode a 6t6 gtilis6e pour le dosage du carbone inorganique et organique des eaux du Boucliercanadien et des carbonates totaux des s6diments.

Received January 22, 197 3

SevmN- techniques exist for the determination ofinorganic carbon in fresh water. Few of these,however, are applicable to the soft waters found inlakes of the Precambrian Shield where concentrationof dissolved inorganic carbon (DIC) are often under100 pmoles per liter. Problems with the widelyused acid titration were discussed by Schindler andHolmgren (1971). The gravimetric technique ofGoldman (1963) and the dialysis concentrationtechnique of Johnson et al. (1970), while effective,require prohibitively large quantities of sampleand are quite time consuming. This work describesa modified gas-chromatographic technique foranalysis of DIC.

Previously reported gas-chromatographic tech-niques suffered from problems arising from thedynamic nature of the sparging systems employed.Swinnerton et al. (1962) reported a techniqueinvolving injection of a small volume of acidifiedsample into a flowing gas stream. CO2 was stripped

Frinted in Canada (J2806)

from the sample by the gas stream and sweptdirectly onto the chromatographic column. Thiswas a nonequilibrium, dynamic system since CO,stripping was not instantaneous. The resultingpeaks were broad and asymmetric, requiring areaintegration for quantitative work. The limit ofdetection for CO2 was about 25 1tw.

An improvement to the above approach waseffected by Williams and Miller (1962), who employ-ed a system that achieved dynamic equilibriumbetween acidified water and helium run counter-currently through a series of rotating Mylar discs.Samples of the equilibrated gas phase were injectedonto the chromatographic column via a sampleloop valve. While this method has been found togive us excellent quantitative data, the need toestablish a dynamic equilibrium for each sampleanaTyzed is consumptive of time (10 min per sample)and sample (500 cm3).

The use of a syringe as a reaction chamber toestablish a static equilibrium of dissolved gasbetween liquid and helium has been reported by

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McAuliffe (1971) for cletermining hydrccarbonsin water and Stainton (1971) for determining mer-cury in tissue. The methcd described below usesa 50-cm3 polypropylene syringe as a reacticnchamber to establish static eouiiibririrn beirveen20 cm3 of sample, 1 cm3 of diinte acid, and, 29cm3 of helium. The analytical system is simple,inexpensive, and provides sensitivity (detecticnlimit I plr CO2) sufficient for most studies of CO2in water. Precision is excellent with a coefficient ofvariation af 0.737o at the 100 trM level. The methodhas been successfully employed to measure DICin fresh water, dissolved organic ca-rbon in freshwater (following a wet oxidation by the methcdof Menzel and Vaccaro (1964)) and to'.al car'oonatesin sediments (following acid digestion at elevatedtemperatures).

Maferials and Me{hodsDrssor,vro IxonclNrc CaneoN

Water samples were collected in 50-cr:,3 polyprcpylenesyringes, directly from a 3Jiter van Dcrn sci::pler.Syringes were capped immediately upon filling and

JOURNAL FISHERIES RESEARCH BOARD OF CANADA. VOL. 30, NO. 10. 1973

transported to the la-bcratory. A11 but 20 cm3 of sampler,.,;as e;<pelled fiorn the syringe for ccnductance, tem-,Dera'iui€, a-ad pil. neasurefflents. Tie remaining 20-crr3sample was ac;cIified r'vith I cr-ri3 of 2n srllphuric acidil:j:cted in':o the sarnple sylinge from a Colnwall re-peaiing syringe via a double ferrralo iuer coupiing. Thesyringe containing the acidified sarnple was then atta-chedto the thiee-way valve shown in Fig. 1 and heliuminjecied at low pressure until the plunger reached the5g-sm3 graduation. The syiinge was quickly cappedand agitated vigorously by hand for 10 sec. If necessary,groups of samples processed to this stage could be leftstanding for several hours while the gas chromatograph(Fisher l{amilton }vlodel 29) was calibrated.

All water used in this study was from a Millipore"Super Q" system. The effluent from this system wastyprcally less than 5 pr"r in CO2 and less than 50 pu indissolved oiganic carbon.

Using the stock bica-rbonate standard (0.8401 g dryNaIJCO:/liter of "Super Q" v'rater), 100 cm3 quantitiesof standards ,,vere piepaied fresh daily to cover the rangefrom 100 to 20G0 pmoles CO2/liter. Blanks and stan-dalds rvere stoied in stoppered 100-cm3 vclumetricflasks iintil useC. Using a 50-crn3 syringe with the plungerfully depressed, 2A crr,j of blank or standard solutionwere drawn up ta-king care not to include bubbles of air

VE NT

CARRiE I? GA5HE i_ tu i d

H E L I U MT R I

TN

I N G T O W

f " n r rn r r " It

LSEE FIC 2 J

HELIUM FOR SAh4PLEP R O C E S S I N G

f*' 1. Analytical system for CO2 determination. A, Detector - thermal conductance type; B, Recorder_ - I mvfull_scale-0.2inches permin; C, Chromatographiccoft-rrnn - 4ft x *inchro - ForopafQ packing; D, "Drierite"drying column; E, Sarnple loop valve; F, Sample loop- 1-5 cm3 depending on sensitivity needed; d, I{elium CO2mixture; H, Acidified sample; J, Syringe plunger; K, Tl.rree-way valve.

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with the liquid. Btanks and standards were acidifiedand partitioned with helium as outlined above forsamples.

A syringe containing the acidified sam 1e partitionedwith heiium was attached to the three-way valve. Withthe three-way valve in position for injection through thesample loop valve, 15 cm3 of the gas phase in the syringewas expelled and the sample loop valve quickly switchedto inject the helium-CO2 mixture onto the chrornato-graphic columas. This procedure was repeafed for blanks,standards, and samples.

Feak height and attenuation required for blanks,standards, and samples were recorded. A scale factor(rrrnoles per liter per chart unit) was calculated fromstandard peak heights corrected for blank. Sample peakheights were simply multiplied by this scale factor to givepmoles CO2 per liter, there being no sample blank.

Drssor-vro OncaNrc C.qnsoN

The procedure outlined is essentially that of l\4enze1and Vaccaro (1964). Modifications have been made toutilize the economy, sensitivity, and linearity of a gas-chromatographic ana.lysis for CO2.

A 50-cm3 sample of water previously passed througha Whatman GF/C filter was added to the strippingtower diagramed in Fig. 2. Helium flow was adjusted toproduce vigorous bubbling through the filter frit and0.1 crn3 of analytical reagent grade perchloric acid6AVa wlw; S.G. 1.54) was added. The acidified samplewas bubbled for 10 min to remove inorganic carbon.The drain tube was then flushed with 5 cm3 of sampleand 25 cm3 of sample run off into a preignited (550 C

t : 2 . 5 c m

3 0 c m

E X T E N S I O N

G L A S S D R A I N A G E P O R T

C LAIV P

FRIT PLAST IC DRAINTUEE TOA M P O U L E S

STOPCOC K

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for 8 hr) glass ampoule containing 0'l g of analyticalreagent grade potassium persulphate. Care was takento avoid percolating the sample through air, the draintube being held beneath the liquid surface in the ampouleduring filling. Tl-re neck of the arnpoule was then sealedwith a propane oxygen flame taking care not to entrapflame combustion gases in the ampoule. Sealed ampouleswere then autoclaved at 121 C for I hr to combust or-ganic matter to CO2. Samples could be stored indefinitelyat this stage.

Samples wet oxidized as above and cooled to roomtemperature were quickly transferred from openedampoules to 50-cm3 polypropylene syringes. A widebore cannula was used to permit rapid transfer of liquidwith minimum degassing. Samples were then analyzedfor CO2 content as outlined previously for inorganiccarbon.

The method of sample transfer from ampoule tosyringe permits loss of any COz in the gas space in theampoule neck. This appears to be a fairly constant 4/sof the CO2 leve1 of the sample. For this reason and asa check on the effectiveness of oxidation it was foundnecessary to calibrate the analyses by treating glucosestandards and Iow carbon water by the same procedureas samples. A scale factor was computed from theglucose standard peak height, corrected for low carbonwater blank. No blank correction was necessary forsamples.

CannoNArE rN SrorrrarNrs

Appropriate quantities of dried samples of calcite(analytical reagent grade CaCO3) and dolomite (99.9%MgCOg'CaCO3) standards were weighed to the nearest10 p! and placed in preignited (550 C for 8 hr) glassampoules. The quantity weighed provided between0.5 and 50 prnoles of carbonate and bjcarbonate carbonper ampoule. Twenty-five cubic centimeters of 1ow carbonwater were then added to the ampoules, followed by50 pliters of concentrated sulphuric acid and the am-poutres sealed immediately. Sealed ampoules wereautoclaved for t hr at 121 C and liberated CO2 deter-mined as for dissolved inorganic carbon. Calibration wasvia calcite and dolomite standards analyzed by the sameprocedure as samples.

Results and Discussion

Drssorvno IxoRcl,Ntc Cnneol'{

A series of six replicates of DIC at the 100-plrlevel was found to have a coemcient of varianceof 0.76%. Sensitivity was found to be about 0.3pmoles CO2 per liter per pv with response beinglinear up to 10,000 pM. The partitioning coemcientof CO2 between sample and helium was measwedto be 1.04. With the ratio of gas to liquid used(29:21), 60Vo of available CO2 was partitionedfrom liquid to gas phase.

Samples held for 4 hr after the 10-sec agitationshowed no detectable loss or gain of DIC from

STAINTON: METHOD FOR DETERMINING DISSOLVED INORGANIC CARBON

+ H E L I U I M I N

Fro, 2. CO2 stripping tower fromMiliipore filtration manifold.

modified 25-mm

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capped syringes. It was therefore possible to batchprocess samples, usually at the rate of one analysisevery 2 min.

Although sensitivity of the method can beenhanced by increasing the sample loop volumeof the gas chromatograph and increasing the ratioof sample liquid to helium, the conditions outlinedabove were quite adequate for analysis of Shieldwaters.

Drssorvpo OnclNrc C.q.nsoN

The method as outlined was tested for precisionusing six replicates of a glucose solution 1000plr in carbon. Sensitivity was of course the sameas for inorganic carbon; however, precisiondeteriorated with a coefficient of variance of 2.3/6.Blanks for the low carbon water used varied from30 to 50 pmoles C/liter with only l-2 prmoles ofthis being a teagent blank. This reagent blankwas ignored in subsequent calculations of dissolvedorganic carbon in samples.

Recovery was tested in two ways, first withaqueous solutions of known organic compoundsand later with laC-labelled algal metabolites andexcretory products.

Results of recovery tests using known compoundsappear in Table I with recoveries expressed relativeto both bicarbonate and glucose standards carriedthrough the same wet oxidation. For the ratherlimited range of compounds tested, recoveriesappear to be acceptable.

To obtain an indication of how the methodwould cope with naturally occurring freshwaterorganics, a solution of 1aC labelled organics wasprepared by growing a culture of Anabaena flos-aqua in a closed system for 15 days with 14C bi-carbonate as the only carbon source, The filtrate

Taert 1. Recovery of carbon from some pure organiccompounds by gas chromatography following wetoxidation. All tests run at the 1000 4rr.r level.

Compound

/6 recoveryrelative to /o recovery

bicarbonate relative to glucosestandard standard

JOURNAL FISHERIES RESEARCH BOARD OF CANADA, VOL, 30, NO. 10. 1973

through a O.22-1t membrane fllter was acidified andactivity measured by liquid scintillation counting.The acidifled filtrate was then bubbled with heliumfor 30 min to remove 1aCO2, and the activity ofthe stripped filtrate measured again, with 64% ofsolution's original activity remaining, presumablyin dissolved organic compounds. The filtrate wasthen wet oxidized at various dilutions as describedabove, bubbled with helium for 30 min and remain-ing activity measured. The amount of activityremaining after this treatment was an indicationof the amount of labelled organic matter notoxidized to COr. At the 7500 prrl carbon level lloof laC activity remained, while at the 1500 pMlevel 0.1/o remained.

These results are not a conclusive demonstrationof oxidation efficiency as the 2,week labelling periodmay not have been sufficient to label the morerefractory compounds present in natural waters.

CnnnoN.trn IN SEDTMENTS

Six replicates of calcium carbonates, dolomite,and dried sediment from Lake Winnipeg treatedas outlined in the method, gave the recoveriesand precisions listed in Table 2. Method specificitywas tested by carrying samples of dried humicextract and acid leached sediment through theanalysis. No detectable breakdown of organiccarbon to CO2 was observed. Apparently the acidconditions and temperature employed are notsufficient to oxidize sediment organics.

The above techniques have been found to beapplicable in many field situations. The smallsample size, low contamination, speed, and sen-sitivity of the analysis have permitted accurateanalyses where it was not possible before. Themethods for dissolved organic and inorganiccarbon allow samples to be taken with a syringefrom sharply stratified lakes or tanks at levelsdeflned to within a few centimeters. The methodfor inorganic carbon has been found useful inestimating benthic primary production by DIC

Tartu 2. Recovery efficiency and precision for gas-chromatographic analysis of sedimentary calcium car-bonate and dolomite.

Nitrilotriacetic acid(as tri sodium salt)

Oxalic acidTartaric acidDextranIJreaSucroseGlucose

9 8 . 495.49 8 . 999.694.49 8 . 399.4

99.09 6 . 099.5

1 0 0 . 39 5 . 098.9

1 0 0 . 0

9 9 . )

101 .22 . 8 %r . 2%

0 .4%

CompoundPrecision (rel. so)

/6 recovery (n:6)

Calcium carbonateDolomiteSediment - Lake

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uptake (Schindler eI al. 1973) and for measuringdiurnal CO2 fluctuations.

Acknowledgments

I thank D. W. Schindler and F. A. J. Armstrongfor critical comments, and R. V. Schmidt for fieldtesting the methods.

Gor,orraaN, C. R. 1963. The measurement of primaryproductivity and limiting factors in fresh water withcarbon-l4, p. 103-113. In M.S. Doty [ed.] Proc. ofthe conference on primary productivity measurements,marine and fresh water, held at University of Hawaii,Aug. 21-Sept. 6, 1.961. U.S. At. Energy Comm.Div. Tech. Inf.

JoHNsoN, M. G., M. F. P. Mrcn.tr,sKr, AND A. E. Cnnrs-TrE. 1970. Determination of low concentrations ofinorganic carbon in lake water. Limnol. Oceanogr.15: 481-487.

McAur,r,rEr, C. 1971. Gas chromatographic deter-mination of solutes by multiple phase equilibrium.Chem. Technol. 1: 46.

r44s

MENzEL, D. W., AND R. F. Vacc.q.no. 1964, Themeasurement of dissolved organic and particulatecarbon in sea water. Limnol. Oceanogr. 9: 138-142.

ScHruonn, D. W., V. E. Fnosr, AND R. V. Scr*tor.1973. Production of epilithiphyton in two lakes ofthe Experimental Lakes Area, northwestern Ontario.J. Fish. Res. Board Can. 30: 1511-1,524.

ScnrNor,En, D. W., .q.ND S. K. HorucruN. 1971.Primary production and phytoplankton in the Ex-perimental Lakes Area, northwestern Ontario, andother low-carbonate waters, and a liquid scintillationmethod for determinin g 1aC activity in photosynthesis.J. Fish. Res. Board Can. 28: 189J01.

SrarNroN, M. P. 1971. Syringe procedures for transferof nanogram quantities of mercury vapor for flamelessatomic absorption spectrophotometry. Anal. Chem.43:625-627.

SwmNunroN, J. W., V. J. LrNNENnou, AND C. H. CHEEK.1.962. Revised sampling procedure for determina'tion of dissolved gases in solution by gas chromato-graphy. Anal. Chem. 34: 1509.

Wrnraus, D. D.,.lNo R. R. Mrlr,nn. 1962. An instru-ment for on-stream stripping and gas chromatographicdetermination of dissolved gases in liquids. Anal.Chem. 34: 675-659.

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