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1528 ANALYTICAL CHEMISTRY, VOL. 50, NO. 11, SEPTEMBER 1978

Automated Separation and Conductimetric Determination ofAmmonia and Dissolved Carbon DioxideRo b e r t M. Car lsonDepartment of Pomology, University of California, Davis, California 956 76

A continuous flow instrument for autom ated determ inatio n ofammonia or dissolved carbon dioxide has been developed.The sample solut ion stream is mix ed with sodium hydroxid efor amm onia determination of perchloric acid for carbo n dioxidedeterm inatio n. Th e ammonia or carbo n dioxide diffuses fromthe sample stream through sil icone rubber hollow f ibers intoa stream of deion ized water. A s the water stream emergesfrom the hollow f ibers, it passes through an electr ical con-duct ivity cell. The conduct ivity response is related to am -monium or carbo n dioxide in the sample. The averag e relativestandard deviation for to tal nitrog en in 39 leaf samples digestedby the Kjeldahl procedu re was 0.66 %. Agreement betweenthe new method and d ist i l lat ion-t it rat ion of Kjeldah l digestswas very good. Reco very of nitrogen for the Bu reau ofStandards orchard leaf reference sample was within onestandard deviat ion of the cert if ied value.

Various procedures for th e determination of amm onia byelect r ical conduct ivi ty me asurem ents have been reported.Hendricks e t al. ( I ) described a vacuum distillation procedurein which th e amm onia was collected in boric or sulfuric acidand determined b y the change in conductivity of the acid. Ina similar procedure, Appleton ( 2 )distilled amm onia into boricacid, diluted the solution to known volume, and determinedammonia from conduct ivi ty changes . Shaw and Stad don ( 3 )used a diffusion cell t o transfer amm onia from the sample tosulfuric acid for its subsequen t determinatio n by conductivity.Fried1 ( 4 ) determ ined amm onia from the ra te of change ofcondu ctivity of a small volume of sulfuric acid as it absorbedamm onia from a sample in a diffusion cell . Sep arat ion ofammo nia by dis t i l la t ion ( 5 ) and gas diffusion (6) have beenemployed in au tomated colorimetric methods .Diffus ion throug h plas t ic tubing has been reported as atechniqu e for separat in g gaseous components of samples .Kollig e t al . (7 ) used silicone rubber tubing in a device forsampling waters for dissolved oxygen, nitrogen, and c arbondioxide determ inations. Scaran o and Calcagno (8)determineddissolved carbon dioxide from pH changes in a bicarbonatesolut ion f lowing through a Teflon tube immersed in thesample. Westover et al . (9) descrihed a sam pling device formass spectrometry th at was based on gas permeation throughsilicone rubb er hollow fibers.Th e work reported h ere was carried out to develop a systemfor automated ammonia determinations in biological samplesafter Kjeldahl digestion. Th e method is based on the transferof ammo nia by diffusion throug h silicone rubber hollow fibersinto a flpwing stream of water, followed by detection byelectrical conductivity. Th e appar atus was also found to beuseful for ammo nia determination in water samples and fordissolved carbon dioxide determinations.

EXPERIMENTALAp pa ra tus . The gas permeation tube (Figure 1)was preparedby attaching an */*-in. .d. polypropylene Y connector t o each endof a 50-cm length of 0.062-in. i.d. small bore polyethylene tubing

with sho rt sections of silicone rubber tubing (1,/,6-in. .d., 3/ls-in.0.d.). Twelve strands of Dow Corning silicone rubber hollow fibers(obtained from Bio-Rad Lab oratories, Richmond, Calif.) wereplaced in the tub e and potte d into one branch of the Y connectoron each end with Dow Corning silicone rubber sealer.Th e electrical conductivity cell is shown schematically in Figure2. Th e well formed with t he v arious sizes of vinyl tubin g wasfilled with styrene casting resin to hold the electrodes of the cellrigidly in place. Thi s constr uction permits placement of theconductivity cell very close to the otitlet of th e hollow fiber bundle.Figure 3 is a block diagram of the complete apparatus.Comp onents used to assemble the apparatus are as follows: ArthurH. Thomas Co., Little Dipper Automatic Sampler, equipped witha timer adjustable from 0.2 to 5 min; Gilson Model HP-4, 4-channel peristaltic pump; Thermonix Model 1480 ConstantTemperature Circulator; Markson Science, Inc. Model 4405Conductivity Analyzer; and Linear In strum ents C orp. Model 254Strip Chart Recorder.The sample and NaOH (o r HC10J streams are mixed in amicromixing device constructed as described by Gugger andMozersky ( 1 0 ) . Air is injected into th e mixed sample stream jus tas it exits the micromixer. Th e stream then enters the ther-mostated water bath and passes through a 50-cm coil of 2-mn id.,1-mm wall glass tubing to bring th e sample to bath te mp erature .It the n passes through the tu be containing the hollow fibers andis discharged t,o waste.Dionized distilled water is pumped through a small column ofmixed bed resin for furthe r polishing. Thi s small column wasconstructed from the polypropylene barrel of a 6-mL disposiblesyringe. The resin is supported in the column by disks of porouspolyethylene. As the water strea m leaves this column, it passesthrough a 25-mm Swinnex filter holder with a Millipore type AAmembrane to remove any particulate matter th at might clog thehollow fibers. The water stream then en ters the ther mostatedbath , passes through the degassing u nit, another small, mixed beddeionizing column (constructed as above but with a 1-mL syringebarrel), through the hollow fibers, the conductiviy cell, and isdischarged to waste. Th e degassing unit consists of a siliconerubber hollow fiber m initube (Bio-Rad Laboratories) with theouter chamber connected to a house vacuum system throu gh a1-L vacuum flask ballast an d a check valve. Inclusion of thedegasser prevents air bubble formation in the hollow fibers andthe conductivity cell when the bath is operated at elevatedtemp erature . The sample, reagent, and water transmission lineswere cut from 0 .042-in. i.d. polyethylene tubing. Connections weremade with silicone rubber or vinyl tubing.Re age nts. All reagents were prepared from analytical reagentgrade chemicals. Samples were mixed with 1.5M NaOH cotaining6 g Na2EDTA.2H20/L. The EDTA was added to preventprecipitation of polyvalent metal hydroxides and carbonates.Carbon dioxide determinations were carried out by mixing thesample with 1 M HC104.Op era tin g Proce du re. The start-up procedure consisted ofpumping water throug h the reagent and sample lines while thetemperature b ath stabilized (10 to 15 min.). These lines were alsoflushed with water fo r 5 min at instrument shutdown. When notin use, the degassing unit was disconnected from the vacuum lineto pre vent loss of water from the hollow fibers.Kj eld ah l Digestion. Plant tissue samples, 250-mg aliquots,were digested in calibrated 75-mL digestion tubes in a blockdigestor. Th e digestion mixtu re was 5 mL HZSO,, 3.5 g KzS04,and 40 mg C uS0 4. One selenized boiling chip was added to eachtube. The samples were heated to 370 "C and held at this

0003-2700/78/0350-1528$01 . OO / O C 1978 American Chemical Society

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ANALYTICAL CHEMISTRY, VOL. 50, NO. 11, SEPTEMBER 1978 1529F I B E R S P O T T E D W I T HS I L I C O N E R U B B E RL v

T U B I N Gr( 0L Y P R O P Y L E N E

C O N N ECTORY "

Flgure 1. Schematic diagram of silicone rubber hollow fiber permeationunit

Figure 2. Schematic diagram of electrical conductivity cell. A, B, C,D are vinyl tubing: (A ) X 1 / 4 , (C) /4 X 3/8 , (D) 3/ aX '/* nches (i.d. X o.d.), (E) 6-cm section of 14 gauge stainless steeltubing, (F) leads to conductivity meter, (G ) 0.8-mm diameter stainlesssteel wireX ' I 8 , B)

L aE l ON l Z l YO VACUUMCOLUMN

Flgure 3. Block diagram of complete instrumenttemperature for 2 h. After cooling, they were diluted t o 7 5 mLand mixed.

R E S U L T S AND DISCUSSIONEven though t he surface area of the f iber bundle is large(es t imated to b e 47 cm2 for a 50-cm long uni t containing 12fibers), the p umping rate required to allow the water passingthrough the fibers to reach equilibrium with the sample is tooslow for rapid processing of samples. T he system depe ndson a steady-state transfer of gas rath er tha n equilibration ofthe water s t ream with the sample s tream. Th e diffus ion ofgas through the fiber walls is temperature-dependent as is theelectrical conductivity of th e resulting amm onium hydroxide

or carbonic acid solut ions. Th e overal l tempe rature de-

-- ~~- - ~.~3 M I Nt--$- ~... ~--e- ~+ ~ - ~-L-

~ ...~- - ~ ~ - ~ -Figure 4. Typical recorder trace for standards and Kjeldahl nitrogensamples. Standards indicated by millimolar concentration of ammonium,samples indicated by numberpendence was determined by noting the response to anammonium s tandard at various temperatures between 25 an d50 "C . Th e response increased by approximately 4% perdegree. Hence, a bath th at wll maintain temperature constantto i .01 "C provides adequate control of this variable.An estimate of the e xten t of gas transfer was obtained bycollecting the water passing through th e fibers while aM NH4Cl was pum ped through the un i t a t 4 .98 mL /minmixed with 1.5 M N a O H a t 1.17 mL /min . Th e flow rate ofwater through the f ibers was 0.98 mL/min and the bathtemperature was 35 "C. The water from the f ibers wascollected in a small volume of 1 M HC10, to prevent vola-til ization losses. Th is solution was the n run through t heins t rument as a sample wi th appropria te s tandards to de-termine th e amm onium concentrat ion, which was found t obe 2.6% of the amm onium concentration in the NH 4Cl/NaO Hstream. For ad aptat ion to samples wi th very low levels ofamm onium , the extent of gas transfer could be increased byemploying a uni t wi th more and/or longer f ibers and byoperat ing a t higher temperatures . Decreas ing the pump ingrate of water through the fibers will also increase the con-centration of ammonia in the solution reaching the con-ductivity cell.In addi t ion to ma in ta in ing cons tan t t empera ture , t hepump ing rates mu st be constant (especially th at of the waterpassing through the f ibers) to obtain s table response. Th eresponse t ime depends on the ra te a t which sample andreagent are pum ped over the f ibers and on th e ra te water ispumped through the f ibers. Typical sampling time used inthis work was 90 s with an addi t ional 10 s required for thesampler to ra ise the probe a nd advance to the next sample.Air is aspirated between sam ples. This provides a convenientspike in the recorder trace th at separates samp les of similarcomposition. Figure 4 shows a typical recorder trace.Th e effect of th e total concentration of salts in the samplestream is an additional variable that requires attention. Th e

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1530 ANALYTICAL CHEMISTRY, VOL. 50, NO. 11, SEPTEMBER 1978Table I.Determination with Distillation-Titration forDigested Leaf Samples

Comparison of Instrumen tal Nitrogen

N , 96distillation-sample instrumen tal t i tration

apple 2.21 2.24pear 2.27 2.26peach 2.55 2.55almond 1.62 1.62mixed species 2.17 2.14prune 2.70 2.69prune 2.94 2.93prune 2.25 2.29prune 2.11 2.10prune 2.33 2.34

partial pressure of ammonia or carbon dioxide will dependon the total salt load. For water samples, the total con-centration will be dom inated by th e Na OH or HC104 mixedwith th e sam ple, so samp le to samp le variation will be small.For digested samples, the sodium sulfate formed in theneutralization of sulfuric acid an d the potassium sulfate inthe digestion mixture will be major components of the saltload. Stan dard s for these determinat ions should containsulfuric acid an d potassium sulfate a t concentrations similarto those of the samples. Th e amoun t of potassium sulfaterequired ca n be calculated b ut some sulfuric acid is consumedin the digestion so the acid concen tration in a few samplesshould be determined to es tabl ish the amount of acid to beadded to th e s tandards. As the am ount of acid consumed inth e digestion will vary som ewh at, the effect of variable acidconcentration was determined. Amm onium chloride standard swere prepared with 30, 60, and 90 mL sulfuric acid/L . In-s t rument response decreased 2.5% as acid concentrationincreased from 30 to 90 mL /L. Th e acid concentration fordigested samples ranged from 50.5 to 55.5 mL /L. The effectof this variation is insignificant. Sta nd ard s were prepared tocontain th e average acid concentration found in the samplesto avoid systematic errors.Most of the precision and accuracy determinations weredone with plant tissue samples that had been carried throughthe Kjeldahl digestion procedure. Precision was determinedby running 39 digested plant leaf samples through the in-s t rum ent in a di fferent random order on each of 5 differentdays. These determinations were made on the same digestedsolutions in order to eliminate variability in th e weighing anddigestion of the samples. Th e average relative standarddeviation for the 39 samples was 0.66%. Th e relative standarddeviation exceeded 1% for only 2 of the samples. Th eprecision of the instrument is quite good.Accuracy was determined by comparing the nitrogen foundin the National Bureau of Standards Reference Material 1571(Orchard Leaves) with the certified value an d by com paringthe ni t rogen found with the ins t rument wi th tha t found bydistil lation and titration for ten samples of deciduous fruittree leaves. Th e mean nitrogen concentration an d standarddeviation found for the Bureau of Stand ards sample was 2.72f 0.02% (11determ ination s). Th e certified value of thissam ple is 2.76 f 0.05%. Th e results comparing instrume ntalwith distil lation-titration methods are tabulated in Table I.Th e determinations by the two method s were carried out onthe same digested samples to eliminate variability in theweighing and digestion of the samples. The re is no significantdifference between the methods.The usefulness of the apparatus for determination ofamm onium in water samples depends on its response to lowconcentration of amm onium. Th e lower limit of response isdetermined by the changes in electrical conductivity as

Flgure 5. Calculated specific conductivity vs. ammonia concentrationTable 11.Carbonate plus Bicarbonate with Results from Titration

Comparison of Instrumental Determination ofC 0 , ' - + HCO,-, equiv/L

sample instrumental t i trationdrain water 1 6.69 6.82 5.65 5.43 4.56 4.84 6.35 6.1well water 1 7.50 7.22 7.91 7.6

ammonia dissolves in water. Th e conductivity is a functionof the concentrations of amm onium , hydroxide, and hydrogenions. Addition of amm onia decreases hydrogen an d increasesammonium a nd hydroxide ion concentration s. H ydrogen ismuch more mobi le than ammonium ion so the change inconductivity with the initial addition of ammon ia is very small.This can be shown by calculating the specific conductivityfrom the limiting ionic mobilit ies of th e three ions and th eammonia-water equilibrium. Figure 5 shows the calculatedconductivity as a function of the square root of ammoniaconcentrations a t 25 "C. Th e limiting ionic mobilities usedwer e: H', 350; OH-, 198; and NH4+,73.4. T he dissociationconstant for ammonium hydroxide was taken as 1.77 XTh e figure shows the lag in response with the initial additionof amm onia. Calibration curves obtained with dilute am-monium standards were consistent with the calculated re-sponse. When th e sample, reagent, and water pumping ra teswere 4.98, 1.17,and 0.96 mL /m in, the useful lower limit wasabout 10-5M NH4+ n the sample. This is adequate for mostroutine w ater analysis.Conversion of the app aratus to determin e total dissolvedcarbon dioxide is accomplished by switching th e reagen t linefrom sodium hydroxide to an approp ria te acid. Perchloricacid was used in this stud y. Sulfuric acid should work equallywell. Suitability for dissolved carbon dioxide dete rmin ation swas tested by comparing results for six drain water and wellwater samples with carbonate plus bicarbonate concentrationsdetermined by titration. Conce ntrations of carbonate andbicar bon ate were com pute d from total r!issolved carb on di-oxide determined by the instrum ent, the dissociation constantsof carbonic acid, and th e pH of the water samples. Stan dard swere prepared from sodium bicarbonate. Th e results aretabulated in Table 11 The agreement between the methods

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ANALYTICAL CHEMISTRY, VOL. 50, NO. 1'1, SEPTEMBER 1978 1531for nitrate determina tions by passing the sample stream overa bed of Devarda alloy to convert nitrate to ammonia in aman ner similar to th at described by Mertens e t al . (11).The method described here has some advantages overmethods current ly in use for ammonia determinat ion.Com pared with th e distillation-titration procedu re, it is faster,easier to autom ate, and can be used with smaller samples. Th ereagents are simpler than those used in colorimetric proceduresand the method will tolerate colored or turbid samples whichcould not be run colorimetrically without pretreatment.Sam ples with high concen trations of solutes can be analyzedif s t and ards contain approximately the same concentrationof solutes. Th e gas sensing ammonia electro de will no t toleratehigh solute concentrations. Electronic drift is muc h less th attha t encountered with the ammonia e lect rode. Th e advan-tages of this new method should make it an attractive al-ternat ive to the methods current ly in use.

LITERATURE CITED(1) R. H.Herdricks,M. D. homas, M. Stout, and B. Tolman, I&. Eng. Chem.,Anal. Ed., 5 , 23-26 (1942).(2) L. Appieton, Chemist-Analyst, 2, 4-7 (1953).(3) J . Shaw and B. W. Staddon, J . Exp. Biol., 35 , 85-95 (1958)(4) F. E. Friedl, Anal. Biochem., 48 , 300-306 (1972).(5 ) J . Keay, and P. M. A. Menage, Analyst(London), 94 , 895-899 (1969).(6 ) Technicon Corporation, Tarrytown, N.Y. , Industrial Method No. 330-74A.(7) H. . Kollig, J . W. Falco. and F . E. Stancil. J r.. Environ. Sci. Techno/.,

9 , 957-960 (1975).(8 ) E. Scarano and C. Calcagno, Ana l . Chem., 47 , 1055-1065 (1975).(9) L. B . Westover, J . C. Tou, and J . H. Mark, Anal. Chem..46 , 568-571(1974).(10) R. E. Gugger and S.M. Mozersky, Anal. Chem., 45 , 1575-1576 (1973).(1 1) J .M e n s , P. Vandenwinkel, and D. .Massart,AM/. Chem.,47 , 522-526(1975).

i s adequa te fo r rout ine water analys is .Volatile acids or bases that would di ffuse through thesilicone fibers will interfere with carbon dioxide or ammoniumdetermination s. Nitrate, chloride, and sulfate salts producedno response when th e instrum ent was set up for carbon dioxidedeterminat ions . Acetate did produce a response and wouldinterfere. Volatile amines are the most significant inter-ferences in ammonium determinations. A test of interferencefrom methylamine and dimethylamine showed a greaterinstrument response to these compounds than to ammoniumsolutions of the same concentration . Samp le color an dturb idi ty have no effect on performance of th e ins t rument .Th e prototype ins t rument has required l i t t le maintenancebeyond periodic replacement of the pu mp tubes. Th e hollowfiber unit shows no signs of deterioration after 24 months ofuse. Th e electrical conduc tivity detection system requiresmuc h less e lect r ical shielding th an potent iometr ic devices.The re is no detectable drift in the baseline after a 5- to 10-minwarm-up period. Th e maximum d rift in response to standardsobserved was abou t 1% over a 3-h period. Segm enting thesample s t ream with a i r shortens the response t ime b ut theinstrument can be operated without air injection if slightlylonger sampling times are used. Th ere is no need for de-bubbl ing the sample s t ream. Th e ins t rument is readi lyadap ted to di fferent concentrat ion ranges by changing thesensitivity of the condu ctivity meter or by changing the sizeof the sam ple and reagent pum p tubes .Preliminary observations indicate additional applicationsof the instrument. Adaptation t o continu ous sampling shouldpresent no problem. I t responds to a tmospheric carbon di-oxide when air is pum ped in place of sample an d reagent soit may have use in gas analysis. It may be possible to use it RECEIVEDor review April 11,1798. Accepted Jun e 23,1978.

Membrane Electrode Measurement of Lysozyme Enzyme UsingLiving Bacterial CellsPaul D'Oraz io ,M. E. M e y e r h o f f , an d G. A. Re c h n i t z 'Department of Chemistry, University of Delaware, Newark, Delaware 19711

Llvlng bacter i al cells of the strain Micrococcus lysodeiktlcusare used as s ubstrate for th e determinat ion of lysozymeact lvlty. The cells are loaded with a marker ion which Isreleased through the act ion of lysozyme upon the cell wall.The rate of ion release is monitored with a highly select ivememb rane electrode and is readily related to the concentrationof enzym e present. The proposed method has excellentsensitlvlty and offers advantages of precision and convenienceover previous turbidimetr ic methods.

Recent ly it has been shown th at l iving bacterial cells canbe used in conjunction with gas sensing electrodes to formbioselective sen sors (1-3) . So used, these cells are effectivelyserving as biocatalys ts a t or near the e lect rode surface.Similarly, othe r vesicles such as sheep red blood cell ghostsloaded with e lect roact ive marker , have been employed asanalytical reagents for the determination of complement andant ibodies by coupl ing the resul tant lysing act ion to an ap-propriate ion selective electrode ( 4 ) . We now have extended

this app roach by loading living bacterial cells with marker andusing these cells as a reagent for the measurement of anantimicrobial enzyme such as lysozyme.Lysozyme (EC 3.2.1.17) catalyzes the h ydrolysis of cell wallsof several Gram-positive bacteria, with maximu m activitytoward Micrococcus lyso deikticus. Th e enzyme acts uponmucopolymers of these walls by breaking p - (1-4)-glycosidicl inkages between al ternat ing N-acetylmuramic acid andN-acetylglucosamine residues ( 5 ) . Action of the enzyme inan osmotically protective medium results in formation ofprotoplasts which are intact living cells having a cell mem branebu t no cell wall (6). However, enzyme action in a hypoton icmedium resul ts in osmotic rupture of the membrane andrelease of the cytoplasmic material.Assays of lysozyme in clinical and research labo ratories ar emost of ten carr ied out by a turbidimetr ic method, wheredecreases in absorbance of a M . lysodeihticus cell suspen sionare measured with t ime a t 450 or 540 nm (7). T h e m e t h odsuffers from a lack of precision and other problems arise fromturbidi ty in the unknown sample. More recently immuno-chemical techniques have been suggested but results, thu s far,0003-2700/78/0350-1531$01.00/0 1978 American Chemical Society