166 r - Analyst, March, 1968, Vol. 93, @@. 166-172 Recorder Automated Procedure for the Simultaneous Determination of Phosphorus and Nitrogen in Plant Tissue BY W. D. BASSON, D. A. STANTON AND R. G. BOHMER (South African Co-op Citrus Exchange, P.O. Box 14105, Lyttelton, Pretoria, South Africa) An automated technique for determining phosphorus in plant tissue in an aliquot of the acid-digested sample issuing from the AutoAnalyzer Kjeldahl Analyzer digestor unit, by an adaptation of the molybdovanado- phosphoric yellow-colour procedure, is described. Optimum reaction condi- tions have been established, and results indicate that the phosphorus deter- mination can be made within the limits of accuracy normally acceptable for similar determinations. I N this laboratory, Technicon AutoAnalyzer instrumental systems are used for determining nitrogen and phosphorus in plant tissue.Nitrogen is determined with the automated K j eldahl Analyzer,l and phosphorus by the molybdovanadophosphoric yellow-colour colori- metric procedure after dry ashing. If, however, the two systems could be coupled and the determinations made simultaneously, the rate of obtaining analytical results would be increased and a considerable saving in manpower made possible. This paper describes the work carried out to develop a technique that could be used to determine phosphorus in an aliquot of the acid-digested sample issuing from the Auto- Analyzer digestor unit. For this purpose, the molybdovanadophosphoric yellow method developed by Kitson and Mellon2 was decided upon for adaptation, because of its simplicity and relative freedom from interference by various anions and cations.I I I mixing coil 0.8 ml/min Ammonium mixing 2.5 ml/min Ammonium rwaster I I I 1 Time delay Colorimeter coi I 410 mji, 15-mm flow cell molybdate vanadate Fig. 1. Flow diagram of apparatus for flow system I 0 SAC and the authors.BASSON, STANTON AND BOHMER 167 This study was based on the assumption that no volatilisation of phosphorus compounds occurred3~4~5~6 during the digestion stage, or, alternatively, that if it did occur, it would be of little consequence because of the identical conditions to which standards and unknowns were subjected. Subsequent comparisons showed that the values obtained by the technique developed and conventional methods are in good agreement.EXPERIMENTAL Studies to establish reactant conditions at different concentrations were carried out with the simplified flow system shown in Fig. 1. This obviated the comparatively lengthy step of passing samples through the digestor unit. Subsequently, the flow system shown in Fig. 2 was used to determine the phosphorus content of the plant digest issuing from the digestor unit. 6 ml/min Digestion mixer rnin Mercury sulphate in Dilution water rnin Dilution water determination determination M EDTA solution Ammonium molybdate Ammonium vanadate Colorimeter 410 mp, 15-mm flow cell Fig. 2. Flow diagram of apparatus for flow system I1 In both of the flow systems the ammonium vanadate and ammonium molybdate solutions are introduced separately into the system. Initially the determination was attempted with a system similar to that developed by Varley,' in which the solutions are pre-mixed during preparation and delivered through one tube into the system.With such a system, phosphorus determinations are neither accurate nor reproducible, and a constant drift in the base-line was observed during a 3 to 4-hour period. Ferretti and Hoffman8 observed a similar drift, and attributed this to incomplete clearing of the flow cuvette between samples. They overcame the problem by devising a flow scheme whereby additional water was pumped into the system between sampling. With flow system I, it was found that introducing the vanadate and molybdate reagents separately into the flow system effected a more satisfactory and less complicated solution.The appearance of the AutoAnalyzer chart recording when the reagents are introduced separately is shown in Fig. 3. No base-line is discernible. When flow system I1 was coupled to the digestor system, base-line drift could not be completely eliminated by introducing the vanadate and molybdate reagents separately into the flow system. (This is probably due to pump surging and incomplete clearing of the flow cuvette because of the high salt content prevailing.) By introducing standard phosphorus samples more often in any series of determinations, this effect can, however, be effectively controlled.168 BASSON et al. : AUTOMATED PROCEDURE FOR THE SIMULTANEOUS [AnaZyst, Vol. 93 L Reagent base-line Fig. 3. The AutoAnalyzer chart recording for the separate introduction of the ammonium vanadate and am- monium molybdate reagents; numbered peaks are standards in p.p.m.of phosphorus REAGENTS FOR FLOW SYSTEM I- Ammonium metavanadate solutions-Prepare a series of solutions containing 0.025, 0.05, 0-15, 0-20, 0.25, 0.30, 0.35 and 0.40 g of analytical-reagent grade ammonium vanadate in 1 litre of distilled water. Ammonium heptamolybdate solutions-Prepare a series of solutions containing, 15, 25, 30, 35, 45, 55, 60 and 70 g of analytical-reagent grade ammonium heptamolybdate in 1 litre of distilled water. Standard PhosPhorus solutions (i)-Prepare a series of five standard solutions containing 10, 20, 40, 80 and 100 p.p.m. of phosphorus from analytical-reagent grade sodium dihydrogen orthophosphate.Standard phosphorus solutions (&)-Prepare a series of fourteen solutions each containing 40 p.p.m. of phosphorus and volumes of hydrochloric acid to give concentrations of 0.04, 0-09, 0.13, 0.17, 0.22, 0.27, 0.31, 0.35, 0.40, 0.44, 0.53, 0.62, 0.71 and 0-82 M when diluted to 100 ml with distilled water. REAGENTS FOR FLOW SYSTEM 11- On the basis of results described in the first section of the Results and discussion, the following vanadate and molybdate concentrations are used. Ammonium metauanadate solution-Dissolve 4 g of ammonium metavanadate in 1 litre of distilled water. Ammonium heptamoly bdate solution-Dissolve 70 g of analytical-reagent grade ammonium heptamolybdate in 1 litre of distilled water. Digestion mixture-Dissolve 6 g of selenium dioxide in a little water and add 1.8 litres of concentrated sulphuric acid, followed by 40 ml of 70 per cent.perchloric acid. Mercury(I1) sulphate solution-Dissolve 35 g of mercury(I1) sulphate in 1 litre of a 10 per cent. sulphuric acid solution. Solution A-Slowly add 910 ml of the digestion mixture to a flask containing 845 ml of distilled water, followed by 66ml of the 3.5 per cent. mercury(I1) sulphate solution and 170 ml of concentrated sulphuric acid. Standard phosphorus solutions (i) and (ii)-To determine the combined effects of the mercury( 11) sulphate, selenium dioxide and perchloric acid on the determination, the following two series of standard solutions containing 10,20,40 and 60 p.p.m. of phosphorus, respectively, are prepared. (i) Sufficient stock solution is placed in a 100-ml calibrated flask to obtain the desired phosphorus concentrations, water isadded to give a total volume of 11.40 ml and this solution diluted to 100ml with solution A.This solution represents conditions after completion ofMarch, 19681 169 the digestion stage as far as acidity, perchloric acid, selenium dioxide and mercury(I1) sulphate concentrations are concerned. (ii) Sufficient stock solution is placed in a 100-ml calibrated flask to obtain the desired phosphorus concentrations, and sulphuric acid is added to obtain a solution about 8 M with regard to sulphuric acid. SAMPLE PREPARATION FOR FLOW SYSTEM I- Weigh 3 g of a dried-leaf sample into a flat-bottomed silica dish and place it in a furnace for 24- hours at 490" C. Cool the dish, add 5 ml of concentrated hydrochloric acid and evaporate the mixture to dryness on a water-bath; continue heating for half an hour.Add 5 ml of concentrated nitric acid, followed by 10 ml of distilled water and warm the solution for a few minutes on a water-bath to aid dissolution. Filter into a 100-ml calibrated flask, wash with warm water and dilute to volume. DETERMINATION OF PHOSPHORUS AND NITROGEN IN PLANT TISSUE SAMPLE PREPARATION FOR FLOW SYSTEM II- Weigh 0.5 g of plant material into a 50-ml Pyrex beaker. Introduce 10 ml of concen- trated sulphuric acid, followed by the slow addition of 15 ml of distilled water. Cover the beaker with a watch-glass and allow it to stand for 20 minutes. This suspension is then placed into the sampler cups for the determination of phosphorus.RESULTS AND DISCUSSION ESTABLISHMENT OF REACTANT CONDITIONS- The influence of variation in the total acid concentration (sample @us vanadate solution) and in ammonium molybdate concentrations, on the colour development, when using flow system I with a standard phosphorus solution containing 40 p.p.m. of phosphorus and main- taining the ammonium vanadate concentration constant at 0.50 g per litre, is shown in Fig. 4. 1 0.1 0 2 0.3 0.4 0.5 0% 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 Total acid concentration (rnolarity) in flow cell Fig. 4. The effect of molybdate concentration on the optical density of a 40 p.p.m. phosphorus solution with 0-50 g per litre of ammonium vanadate and different acid concentrations: curve A, 55 g ; curve B, 45 g ; curve C, 35 g ; curve D, 25 g; and curve E, 15 g of ammonium molybdate per litre For all molybdate concentrations a sharp increase in optical density is observed as the acid concentration decreases below 0.3 M, this increase being less significant at lower molybdate concentrations.For molybdate concentrations less than 25 g per litre, the optical density is observed to decrease as the acid concentration increases above 0.7 M. This effect is not manifested at molybdate concentrations above 25 g per litre. The results obtained by maintaining the ammonium molybdate concentration constant at 55 g per litre, while varying the vanadate and acid concentrations for a series of 40 p.p.m.170 BASSON et al. : AUTOMATED PROCEDURE FOR THE SIMULTANEOUS [Amlyst, Vol. 93 of phosphorus solutions, are shown in Fig.5. A sharp decrease in optical density is observed at the higher acid concentrations for ammonium vanadate concentrations of less than 0.60 g per litre. This effect becomes more pronounced as the vanadate concentration decreases. A .. ---(I-*- 0-*-0--- 0 -- - - -- - -‘-“--xTDc 7 LE 1 1 1 l l l l I l l l l l l l l 1 1 1 1 0 1 0 2 030.40.5 0.6 0.7 0 8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1-7 1’8 1.9 Total acid concentration (molarity) in flow cell Fig. 5. The effect of vanadate concentration on the optical density of a 40 p.p.m. phosphorus solution with 55 g per litre of ammonium molybdate and different acid concentrations: curve A, 1-20 g ; curve B, 0.80 g; curve C, 0.60 g; curve D, 0.40 g; and curve E, 0.20 g of ammonium vanadate per litre We concluded from these results that, when using flow system I, a final acid concen- tration of about 0.7 M , and ammonium vanadate and ammonium molybdate concentrations of 1-00 and 50 g per litre, respectively, would provide the most satisfactory results.Leaf samples were now prepared as previously described for flow system I, and the solutions obtained used to test the reproducibility of peak heights. The results for three samples, viz., A, B and C , are shown in Fig. 3. TABLE I THE PHOSPHORUS CONTENT OF PLANT-TISSUE SAMPLES DETERMINED BY VARIOUS PROCEDURES Phosphorus, per cent. Laboratory reference number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 r Acid extract of dry-ashed sample, flow system I 0.124 0-106 0.087 0-123 0.104 0.114 0.164 0.145 0-126 0.109 0.126 0.137 0.105 0.089 0.115 0.164 0.192 0.124 0.216 0.085 0.234 0.106 0.086 Sample digested on AutoAnalyzer, flow system I1 0.131 0.1 12 0.093 0.130 0.113 0.111 0.167 0.138 0.119 0.114 0.134 0.126 0.116 0.094 0.123 0.144 0.183 0.1 18 0.206 0.084 0.216 0.1 13 0.094 Acid extract of dry-ashed sample, kanual molybdovanadophosphoric yellow colorimetric procedure 0.133 0.103 0.092 0.124 0.108 0.119 0.174 0.144 0.123 0.102 0-124 0.132 0.102 0.082 0.125 0.134 0.175 0-132 0.214 0.076 0-224 0.102 0.083March, 19681 17 1 Identical peak heights for each duplicated standard or sample were obtained, and a constant return to the original reagent base-line between samples or standards over the range 0 to 80 p.p.m.is also observed. The phosphorus values obtained for a series of plant- tissue samples are compared with the values obtained by the manual molybdophosphoric yellow method,!' in Table I.It will be observed that the results compare favourably. DETERMINATION OF PHOSPHORUS AND NITROGEN IN PLANT TISSUE DETERMINATION OF PHOSPHORUS CONTENT IN PLANT MATERIAL AFTER DIGESTION ON THE The acid digest issuing from the digestor unit is about 8 M. It is, therefore, necessary to neutralise the digest substantially to obtain the desired acid concentration previously decided upon. In order to investigate the feasibility of such a step, the flow system shown in Fig. 2, but uncoupled from the digestor unit, was used. Ammonium molybdate and ammonium vanadate concentrations were kept constant and the sodium hydroxide concen- tration was varied.At sodium hydroxide concentrations below 280 g per litre, the results were non-repro- ducible and irregularities in the read-out curves occurred at the peak heights, arising from the high acid concentration still prevailing. At concentrations higher than 350 g per litre, a low degree of reproducibility was obtained, and blockages occurred as a result of crystal- lisation from the standard sodium sulphate solution. A sodium hydroxide concentration of 300 g per litre gave the most constant and reproducible results (Fig. 6). AUTOANALYZER DIGESTOR SYSTEM- I I I I I 1 I 225 250 275 300 325 350 3; Concentration of sodium hydroxide, g per litre Fig. 6. The effect of sodium hydroxide con- centration on the optical density a t constant vanadate and molybdate concentrations : curve A, 30 p.p.m.; curve B, 20 p.p.m. ; and curve C, 10 p.p.m. of phosphorus solution In the digestion of plant tissue for the nitrogen determination, mercury sulphate, selenium dioxide, perchloric and sulphuric acids are used. To determine the combined effect of these substances on the phosphorus determination, the two series of standard phosphorus solutions prepared under Reagents for use with flow system I1 were compared. No difference in the percentage transmission for equivalent phosphorus concentrations was observed. This indicates that the perchloric acid, selenium dioxide and mercury sulphate used in the digestion stage had no effect on the determination. Flow system 11, coupled to the AutoAnalyzer digestor system, was then used to digest and determine the phosphorus content of plant-tissue samples suspended in sulphuric acid (Fig.3), as previously described. Initially, irregularities occurred in the reagent base-line. This was, however, improved by substituting a 0.01 M EDTA solution for the diluting water.172 BASSON, STANTON AND BOHMER Typical recorder charts obtained are shown in Figs. 7 (a) and (b). Fig. 7 (a) shows the reproducibility of replicate determinations on one sample suspension. The reproducibility of determinations on six independently prepared suspensions of sample, H, are shown in Fig. 7 (b). The peak heights V, S, P and T in Fig. 7 (b) represent 0.10, 0.08, 0.13, 0.16 per cent. of phosphorus, respectively. Reagent base-l i ne Fig. 7. Reagent base-l ine (4 (b) Typical chart recordings: (a) the reproducibility of replicate determinations on one sample suspension ; ( b ) the reproducibility of determinations on six independently prepared suspensions : numbered peaks are standards in p.p.m.of phosphorus: peak heights V, S, P and T represent 0.10, 0.08, 0.13 and 0-16 per cent. of phosphorus, respectively The percentage phosphorus values obtained by using this method are compared with values obtained by the manual method in Table I. No significant differences between the results are apparent. CONCLUSION The results obtained in these experiments indicate that phosphorus and nitrogen may be determined simultaneously on the digest issuing from the AutoAnalyzer digestor unit. The phosphorus determination can be made within the limits of accuracy normally acceptable for similar determinations. We gratefully acknowledge the assistance of Mr. N. C. Basson in various aspects of the laboratory work. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. Technicon AutoAnalyzer Methodology Bulletin No. N3a, 1966. Kitson, R. E., and Mellow, M. G., Ind. Engng Chem. Analyt. Edn, 1944, 16, 379. Hillebrand, W. F., Lundell, G. E. F., Bright, H. A., and Hoffman, J. I., “Applied Inorganic Analysis,’’ Second Edition, John Wiley & Sons Inc., New York, 1953, p. 696. Lueck, C. H., and Boltz, D. F., Analyt. Chem., 1956, 28, 1168. Boltz, D. F., Editor, “Colorimetric Determination of Nonmetals,” Interscience Publishers Inc., New York and London, 1958. Calderbank, A., and Turner, J. B., Analyst, 1962, 87, 273. Varley, J. A., Ibid., 1966, 91, 119. Ferretti, R. J., and Hoffman, W. M., J . Ass. 08. Agric. Chem., 1962, 45, 993. Jackson, M. L., “Soil Chemical Analysis,” Constable & Co. Ltd., London, 1962, p. 152. Received Sefitembw 1 lth, 1967