Semi-automated ammonia probe determination of Kjeldahl nitrogen in freshwaters

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SEMI-AUTOMATED AMMONIA PROBE DETERMINATION OF KJELDAHL NITROGEN IN FRESHWATERS R. J, S~vEys Department of A~iculture, Freshwater Biological Investigation Unit. Greenmount Road. Muckamore. Antrim BT41 4PX. Northern Ireland (Receired 9 June 1975)

Abstract--A commercially available ammonia probe was used in an automated continuous flow-through system to determine the ammonia-N concentration in Kjeldahl digests of fresh waters. The method is suitable for samples with KjeldahI-N concentrations greater than 0-1 mg N.I -t. In the 0-1-1'0 mg N.I-t range 10 digests.h-~ can be analysed. Only micro-digestion equipment and sample volumes of 25 ml are required. The ammonia probe with modified filling solution and polytetafluoroethylene membrane is sumciently stable for continuous use throughout a working day. Analyses of Kjeldahl digests of freshwater samples by this method and the distillation-Nesslerisation procedure showed good agreement. More precise results were obtained by the ammonia probe method. The ammonia probe method obviates the steam-distillation operation which is time-consuming and during which errors can be easily introduced by loss or gain of ammonia.

INTRODUCTION

metric method. The distillation-titration procedure The Kjeldahl method is the standard procedure for was replaced by a manual ammonia probe measuredetermining the organic nitrogen plus ammonia con- ment for Kjeldahl digests of soils (Bremner and Tabacentration in freshwater samples. Organic nitrogen tabai. 1972) and meat {Todd. 1973). and by an autocompounds are first digested by acid and catalysts mated ammonia probe measurement for the digests to ammonium salts. The ammonium ion concen- of barley, malt, wort or beer (Buckee, 1974). Distillatration in the digest is then determined by steam- tion of ammonia from the digests prior to the distillation followed by titration for macro-procedures ammonia probe measurement was unnecessary. or by steam-distillation followed by spectrophotoBeckett and Wilson (1974) and Evans and Partridge metric analysis for micro-procedures (Standard (1974) found that the ammonia probe could be used Methods, 1971: Department of the Environment, successfully to manually determine concentrations 1972). These manual methods are time consuming greater than 0-1 mg N.I -~ in discrete freshwater and subject to error by contamination from nitro- samples. Midgley and Torrance (1973) used the probe genous materials in the laboratory atmosphere. in a flow-through system to continuously monitor the Automated digestion and spectrophotometric ammonia concentration in the 0.1-1-0rag N.I -~ analysis procedures have been developed using Tech- range in condensed steam and boiler feedwater. The nicon autoanalyser systems (Kammerer, Rodel, present study describes an automated procedure for Hughes and Lee, 1967: McDaniel, Hamphill and determining the ammonium ion concentration in Donaldson, 1967). To overcome an unstable baseline, Kjeldahl digests of freshwaters using an ammonia lack of sensitivity and irregular peak shape, Harwood probe and a continuous flow-through system. The and Huyser (1970) found that automated ammonia method replaces the manual distillation-Nesslerisadistillation from the digest prior to spectrophoto- tion procedure (Standard Methods, 1971). metric analysis was required. The cost of automated digestion, distillation and spectrophotometric analysis MATERIALSAND METHODS equipment is prohibitive for many laboratories. (a) Kjeldahl digestion procedure Semi-automated procedures where the digestion is Reauents manually performed but the ammonium ion concenAnalytical grade reagents and freshly distilled--deionised tration in the digest is automatically determined water {less than 2 ~4g N.I -t) were used throughout. All reduce the total analysis time considerably, especially glassware was acid-washed and thoroughly rinsed immediately before use. if the distillation step is not necessary. (i) Digestion mixture. One hundred and twenty grams Julian and Kroner (1967) digested the freshwater of anhydrous sodium sulphate and 1-0 g selenium dioxide samples and neutralised the acidic digests manually were dissolved in 600 ml water. Two hundred millilitres before determining the ammonium ion concentration of sulphuric acid (Sp. Gr. 1.84) were slowly added and by an automated phenol-hypochlorite spectrophoto- the mixture diluted to 1-01 with water. 171

172

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liil S~andard ammoni,t solution.s. -% the eflicienc', of con~ersion of organic nitrogen compounds into anamonia during this digestion procedure `'~as assumed to be as good as in any other procedure ammonium chloride aas used for calibration purposes. Ammonium chloride {3.819 g dried at 105:C1 ,.,,as dissolved in 1.0I of ~`'ater to give a standard stock solution containing i000 mg N.I - ~. Dilutions of this solution were freshl? prepared as required to give working standards in the 0.I--1-0 mg N . I - ~ range. {Jail Samples. Unfiltered samples `'~ere digested immediately after collection `'~hen possible. Otherwise samples were stored deep frozen in polythene bottles prior to digestion.

Procedure Twenty five millilitres of sample or standard solution were pipetted into a 50 ml Kjeldahl flask. A small indentation had been made in the rim of the flask to ease subsequent pouring. Five millilitres of digestion mixture were added from a dispenser and 2-3 antibump granules added using forceps. The flask was heated gently on a digestion rack until all the water had boiled off. Then the residue was refluxed for 1 h. When the flask had cooled 5 ml of water were added to prevent sodium sulphate from crystallising out. The digest was transferred to a 25-ml volumetric flask using 3 5-ml water rinses and the volume made up to the mark with water. Then the flask was stoppered and the contents mixed thoroughly. The ammonia-N concentrations in the digested samples and standard solutions were measured by the procedure ot, tlined below. (b) Determination of ammonia in the Kjeldahl diyests

Apparatus li) Ammonia probe. A model 8002-2 ammonia probe (Electronic Instruments Ltd) was used. The principles of operation and construction of the probe have been described previously (Midgley and Torrance, 19721. The probe was equipped with a flow-through cap and modified by fitting a porous polytetrafluoroethylene (PTFE) membrane (Millipore Corporation, Cat. No. FI--ILPOI300), (ii) Mill&air measurement. A Radiometer PHM64 pH/mV meter was used to measure probe potentials to 0-1 mV. The output of the meter was recorded on a Unicam AR25 Series recorder via a simple baseline adjuster as shown in Fig. 1. Different ranges of potential difference could be set to cover a full span on the chart recorder. (iii) Continuous flow apparutu.s. A schematic diagram of the continuous flow apparatus is shown in Fig. 1. A Technicon AAI pump and sampler were used. The sampler was equipped with a glass sample tube to resist the acidic digests. The mixing coil was immersed in a water bath

t30 (_) t<~ d]:,~,lpate the heat e`'oi`'ed ;`'hen the a,:idic digest:~ `'`'ere made alkaline. Since the ammonia probe response is temperature dependent the probe tip `'~as also immersed in the v.ater bath. It ~as tmnece>sar`' to debubNe the airsegmented liquid stream bet\~re passing it through the probe. In the 0 l 1,0 mg N.I ~ range a sampling rate of 10.hr-~ v, ith a sample: wash ratio of l:i v, as suitable. Sample cups of 8 mI capacit.~ ~ere required.

Reorients ~ol p H ~a!jusrin~/ solution. 281 g of sodium h~droxide and 5 g of ethylene diamine tetracetic acid, disodium salt iEDTAI ~ere dissol~ed in 1.01 of water. This solution, when mixed with digest in the ratios shown in Fig. t, contained sufficient sodium hydroxide to neutralise the acid in the digest and to increase the pH to greater than 13. The sodium hydroxide concentration in the solution reaching the probe was approximately 0-2 xl. EDTA releases ammonia chelated to heav~ metals and pre`'ents ~ e precipitation of metal hydroxides Ee.g. Mg [OHI.,] on the membrane surface. (b) IV~tsh solution. 400 ml of digestion mixture and 4.0 ml of 50rag N.I-~ ammonium chloride solution wcrc diluted to 2.0 I. with water, This gave a wash solution of the same acidity and ionic concentration as the digests when they were made up to 25 ml. Ammonium chloride (0.10 mg N.[ -~1 was incorporated in the wash solution so that a baseline response from the probe could be obtained on scale with the chart recorder, and the probe equilibration time bet`'`'een sample and wash would be as rapM as possible. {el Probe lillin~l solution. As the concentration of dissolved salts in the digested samples ,,,,'as high. the ionic concentration of the probe lilling solution had to be increased to pre`"ent osmotic transfer of water through the membrane (Electronic Instruments Ltd. 1974al. Neglecting the salt concentration of the original sample, the concentration of dissolved salts in the measured solution was: li) 0.t4 ,',t sodium sulphate from the digestion mixture. (ii) 0.58 xt sodium sulphate from the neutralisation by sodium hydroxide of sulphuric acid in the digestion mixture, assuming no loss of sulphuric acid during the digestion. (iii) 0-20 .',t excess sodium hydroxide to increase the pH in the measured solution to above 13. The total ionic concentration in the measured solution was therefore 2.56 M. The standard filling solution supplied with the probe was 0-1 r,I ammonium chloride. A modified filling solution was made up by dissolving 0-5349 g ammonium chloride and 11.18 g anhydrous sodium sulphate in I00 m[ of water. To obtain a steady baseline Sampler - Technic~l AAI I0 samples h4ur Sample:wash ratio

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Fig. 1, Continuous flow-through apparatus for determining ammonia-N concentration in Kjeldahl digests of freshwaters.

173

Determination of Kjeldahl nitrogen in freshwaters potential from the probe the sodium sulphate concentration in the modified filling solution had to be decreased to t 1.04 g.(t00 ml}-~. A drop of bromothymol blue indicator was added to the filling solution so that membrane leaks were easily noticeable. td~ Undi~lested standard solutions. Since digestion does not affect ammonium chloride, standard solutions containing digestion mixture were prepared directly for calibration and probe response-time studies. Five millilitres of digestion mixture were pipetted into each 25 mI volumetric flask and. 0.2, 0.4, 0"6. 0-8 and 1-0 mg N.I - t undigested standard solutions were prepared by pipetting 1.0, 2.0. 3.0. 4.0 and 5.0 m[ respectively of 5 mg N.[-1 ammonium chloride solution into the flasks and making up to volume with water. (e) Probe storinfl solution. This solution contained 121.2 g.I- t of sodium sulphate so that its ionic concentration was similar to that of the probe filling solution.

Procedure The probe was assembled as detailed in its instruction manual using a PTFE membrane and modified probe filling solution. The PTFE side. and not the polyethylene backing mesh, of the membrane was placed next to the pH electrode. The required tension on the PTFE membrane was caused by the end of the pH electrode protruding one and one-half turns beyond the end-face of the probe body. When adding the filling solution care was taken to avoid trapping an air bubble between the electrode end and the membrane. The assembled probe was connected into the flowthrough apparatus (Fig. 1). Wash solution, pH adjusting solution and air were pumped continuously. After about 30 rain the probe potential was stable and the analyses of digested standard solutions and digested samples could begin. A close fitting cover prevented atmospheric contamination of the digested solution in the sample cups of the autosampler. When the analyses were finished probe storing solution only was pumped through the system for 5 rain. A calibration curve was prepared by plotting peak heights of the digested standard solutions against the logarithms of their concentrations. The ammonia-N concentrations in the digested samples were obtained from their peak heights and the calibration curve.

Sample cup contamination Atmospheric contamination of the digests while in the sample tray was prevented by the perspex lid which fitted closely over the sample cups. The lid was as effective as covering individual cups with Parafilm sealing tissue (GaUenkamp and Co.) and sampling them with a sharpened probe. A variable response corresponding to 0.10--0-15 mg N.1- t was obtained when the baseline solution containing 0.10 mg N.I -~ was sampled from unwashed disposable polystyrene sample cups. This positive interference was possibly due to a release agent used during the moulding of the cups. Washing with acid, alkali, detergent or water did not completely remove the interference. Acid-washed glass cups had to be used in this laboratory for determining free ammonia concentrations in freshwaters by an automated indophenol-blue procedure. Unfortunately glass cups of 8 ml capacity were not available for the autosampler used in this method. To minimise the interference, disposable cups were washed in detergent and thoroughly rinsed with water immediately before use.

Probe response time amt stability The chart recorder trace for duplicate undigested standards of 0"2, 0-4, 0"6, 0"8 and 1-0 mg N.1 - t concentration is shown in Fig. 2. A sampling time of three minutes was sufficient in this concentration range for the probe to reach a steady potential, and a wash time of 3 min was sufficient for the probe to reach its baseline potential between samples. The baseline potential remained almost constant throughout the day, drifting by less than 0-2 mV.h-t.

Calibration curve In the 0.1-1"0 mg N.I - t concentration range the potential (E) generated by the ammonia probe is related to the ammonia concentration in the measured solution according to the equation: E = Constant - 2"303RT/F log [NH3-N-] {Midgley and Torrance, 1972)

RESULTS AND DISCUSSION

Choice of membrane The membranes supplied with the probe proved unsatisfactory for use with the flow-through cap. After only a few hours the membrane surface became discoloured and the probe response erratic. The membrane did not appear to be sufficiently alkali-resistant. Hawker, Midgley and Torrance (1973) tested a variety of membrane materials and found a PTFE tape to be best. The ammonia probe used by Bailey and Riley (1975) was fitted with a 0.1 mm thick PTFE microporous membrane. In the present study a PTFE membrane with a pore size of 0-5 It and a polyethylene backing was selected. PTFE is naturally hydrophobic and both PTFE and polyethylene are alkali-resistant. The polyethylene backing gives the membrane the necessary rigidity for use in the probe. Membrane life was at least one month, during which time adjustment of the tension between the pH electrode and the membrane was unnecessary.

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decade change in concentration. This slope factor is smaller than the theoretical value (60.1 mV at 30 C) but similar to the values reported b~ Beckett and Wilson/1974i. The slope factor could vary over a period of ~eeks depending on the sensitiviv of the pH electrode. When the factor became less than 55 mV the pH electrode v, as rejuvenated by alternate acid/alkali treatment (Electronic Instruments Ltd, 1974b).

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where R is the gas constant, T is the absolute temperature and F is the Faraday unit. At 30oC equation (1) becomes: E = Constant - 0:0601 log i-NH~-N]

(2)

A plot of the probe potential, measured as mV by the pH/mV meter, or as peak height by the chart recorder, against log [NH3+N] should be linear with a slope of 60-1 mV per decade change in concentration at 30'C. As shown in Fig. 3. the graph of peak height/mV against log [-NH3-N] for duplicate undigested ammonium chloride standards in the 0.1-1-0 mg N.1 -~ range deviated from linearity below 0.4 mg N.1 -~. The slope factor for the linear portion of the graph was 54.8 inV. Deviation from linearity was due to ammonium salt impurities in the digestion mixture reagents. The probe was therefore sensing ammonia from the standard solution plus inpurity ammonia added via the digestion mixture. Midgley and Torrance (1972) reported a similar bias due to the ammonia concentration in the water used to prepare standard solutions. The stated maximum ammonium ion concentration in the sodium sulphate used was 0.0005~.{; (Hopkin and Williams Ltd.). By steam distillation and Nesslerisation the sodium sulphate was found to contain 0-00011",i ammonia-N. Significant ammonium ion impurity could not be detected in any other chemical used. Impurity ammonia added by the sodium sulphate in the digestion mixture to each standard or sample would therefore amount to 0.026 mg N.I -~. The actual ammonia-N concentrations in the undigested standard mlibration solutions were therefore 0.23, 0.43, 0,63, 0.83 and 1.03 mg N . I - t . The ammonia-N concentration in the baseline solution was 0-13 mg N . I - ' . When probe response was plotted against these adjusted concentrations (Fig. 3) the calibration is linear throughout with a slope of 57.1 mV per

When ammonium chloride standards were digested they gave an increased probe response compared to undigested standards. The increase was equivalent to about 0.04 mg N.1-t throughout the concentration range and was due to nitrogen impurities in the sodium sulphate used in the digestion mixture. This discrepancy could be removed by using sodium sulphate recrystallised from water, the insoluble impurities having been filtered out using a glass-fibre paper. Free ammonium ion impurity could also be removed during the recrystal[isation by bubbling ammonia-free air for a few minutes through the hot saturated solution made just alkaline with NaOH. For routine determinations, however, "Analar" sodium sulphate was used without further purification. Since digestion mixture containing impurities was added to the samples and standards alike, the true ammonia-N concentration in the digested samples can be obtained from their probe response and the calibration curve from digested standard solutions.

Compurison of the anm+onia-probe procedure with the distilhltion-Nesslerisation procedure Duplicate determinations of Kjeldahl-N concentration were made for 30 freshwater samples by the digestion-distillation-Nesslerisation procedure (Standard Methods 1971)and by the digestion-ammonia probe procedure. As only micro-digestion equipment was available the volume of sample for digestion was restricted to 25 ml in both methods. The relationship obtained between the results from the two methods was represented by the equation: "Ammonia p r o b e " = 1.002 x "'Distillation-Nesslerisation"- 0-016. (3) The correlation coefficient of the linear regression line was 0-980 and the standard deviation of the slope was 0.039. The null hypothesis procedure (Snedecor and Cochran. 1967) showed that the results given by the ammonia-probe method were not significantly different from the results given by the distillationNesslerisation method at the 95?/0 confidence level. A river water sample with Kjeldahl--nitrogen concentration near the middle of the working range was analysed twenty4our times by each method. More precise results were obtained by the ammonia-probe method (Table 1). Similar analytical precision for ammonia probes was reported by Midgley and Torrance (1972). Beckett and Wilson (1974) and Evans

175

Determination of Kjeldahl nitrogen in freshv,aters Table I. A comparison of the precision of Kjeldahl-N analyses of river v~ater by two methods

Method Ammonia probe DistillationNesslerisation

Number of replicate determinations

Mean concentration of Kjeldahl-N (mg N.I- t)

Standard deviation ling N.I- i)

Coel~cient of variation (ool

24 24

0"43 0"49

0"02 0"06

4 I1

Department of the Environment 119721 7he Analysis of Raw. Potable and Wctsre Wetters, pp. 144--146. H.M.S,O.. London. Electronic Instruments Ltd.. Chertsey. Surrey, (1974a) Application note--Use of the ammonia probe in the determination of tot:t[ organic nitrogen by the Kjeldahl method. Electronic Instruments Ltd.. Chertsey, Surrey, (1974b) ApCONCLUSIONS plication note--Cleaning procedure for ammonia probe pH electrode. The ammonia probe was used for measuring Evans W. H. & Partridge B. F. (1974) Determination of ammonia-N concentration in Kjeldahl digests of ammonia levels in water and wastewater with an ammonia probe. Analyst, Lond. 99, 367-375. freshwaters in the 0.1-1.0 mg N,I - t range. Due to response time and ammonium salts as impurities in Harwood J. E. & Huyser D. J. (19701 Automated Kjeldahl analyses of nitrogenous materials in aqueous solutions. reagents the lower practical limit of the probe was Water Res. 4, 539-545. 0"1 mg N . l - t . The probe was found to be reliable Hawker B. W., Midgley D. & Torrance K. 11973) Potentiometric sensor for determining ammonia in boiler feedand trouble-free only when it was used with PTFE water and condensed steam. Lab. Pract. 24, 724-728. membranes and flow-through cap. Ten samples per hour were analysed by a con- Julian E. C. & Kroner R. C. (1967) Determination of organic nitrogen in water by semi-automatic analysis. tinuous flow system. The procedure was simpler, Technicon Symposium, Automation in Analytical Chemisquicker and gave more precise results than the contry, Vol. l, pp. 542-545. Mediad, White Plains, N.Y. ventional micro--digestion-distillation-Nesslerisation Kammerer P. A, Rodel M. G.. Hughes R. A. & Lee G. F. (1967) Low level Kjeldahl nitrogen determination on procedure. The method is equal!y applicable to the Technicon Autoanalyser. Ent'iron. Sci. TechnoL 1, samples with concentrations greater than 1 mg N.I - t. 340-342. Probe response will then be faster so that sampling Midgley D, & Torrance K. (19721 The determination of ammonia in condensed steam and boiler feed-water with rate could be increased. a potentiometric ammonia probe. An,lyst, Lond. 97, 626-633. Acknowledgements--The author thanks Miss S. Farmer for Midgley D. & Torrance K. (1973) Continuous determinatechnical assistance with the distillation-Nesslerisation tion of ammonia in condensed steam and high-purity procedure. boiler feed-water by using a potentiometric ammonia probe. Analyst. Lond. 98, 217-222. McDaniel W. H., Hemphill R. N. & Donaldson W. T. REFERENCES (1967) Automatic determination of total Kjeldahl nitrogen in estuarine waters. Technicon Symposh~m, AutoBailey P. L. & Riley M. (1975) Performance characteristics nuaion in Analytical Chemistry, Vol. l, pp. 363-367. of gas-sensing membrane probes. Analyst, Lond, 100, Mediad, White Plains, N.Y. 145-156. Beckett M. J. & Wilson A. L. (1974) The manual deter- Snedecor G. W. & Cochran W. G. (1967) Statistical Methods, pp. 91-119. The Iowa State University Press, mination of ammonia in freshwaters using an ammoniaAmes, Iowa. sensitive membrane-electrode. Water Res. 8, 333-340. Bremner J. M. & Tabatabi M. A. (1972) Use of an Standard Methods for the Examination of Water and Wastewater (1971) 13th edn pp. 244--248. American Public ammonia electrode for determination of ammonium in Health Association. New York. Kjeldahl analysis of soils Commun. Soil Sci. PI. Analysis Todd P. M. (1973~ Use of the ammonia electrode for deter3, 159-165. mination of nitrogen in meat products. J. Sci. Fd. Agric. Buckee G. K. (1974) Estimation of nitrogen with an 24, 488. ammonia probe. J. Inst. Brew. 81), 291-294.

and Partridge (1974k The poor precision in the distillation-Nesslerisation procedure was possibly due to errors introduced during the steam distillation operation. Ammonia may be lost when the digest is neutralised, or gained by atmospheric contamination.