Determination of Phosphorus in Milk Powders by Optical Emission Spectrometry with a High Frequency Inductively-Coupled Argon Plasma Source A. M. Gunn, G. F. Kirkbright," and L. N. Ophelm Department of Chemistry, Imperial College, London, S W7,
U.K.
A rapld, dlrect method for the determlnatlon of phosphorus In milk powder solutlons by optical emlsslon spectrometry uslng an Inductively-coupled argon plasma source operated at 27 MHr Is presented. Optlmum condltlons for the determlnatlon have been established and a detectlon limit of 0.1 pg/mL P has been obtained at the phosphorus 213.62 nm line. A study of the preclslon of the technlque has also been carried out and a relatlve standard devlatlon of 2% has been obtained for the complete analytlcal procedure.
The determination of phosphorus in aqueous solution by optical emission spectrometry using an inductively-coupled plasma source (ICP) has been reported by several groups of workers (1-4). The technique has been applied to the determination of phosphorus levels in blood by Greenfield and Smith (5) and Kniseley et al. (6);Kirkbright, Ward, and West (7) have reported its use for the determination of phosphorus in acetic acid extracts of soils. The results of the above studies indicated that the sensitivity attainable by the technique would allow the direct determination of total phosphorus in milk powders. There is a need for a rapid and precise direct method for the determination of phosphorus content of milk products without recourse to destruction of organic matter by wet ashing. As phosphorus is an essential nutritional component in milk, and is present at relatively high levels, it is necessary to achieve good precision in ita determination. The molybdate colorimetric method (AOAC 7.095) is most commonly used for the determination. Inevitably, however, the necessity for wet ashing of samples before development of the absorbing species for solution spectrophotometry results in a lengthy and tedious procedure. In this paper we describe a rapid and convenient emission spectrometric method in which total phosphorus is determined by pneumatic nebulization of aqueous milk powder solutions into the inductively-coupled plasma source. EXPERIMENTAL Apparatus. The instrumental system employed utilized a 2-kW crystal-controlled radiofrequency generator operating at 27 MHz (International Plasma Corporation, Hayward, Calif.) and a 1-m plane grating scanning monochromator (Rank Hilger Ltd., Margate, Kent, U.K.). A demountable plasma torch assembly with tangential argon inlets and sample introduction from a central injector tube was used in this work. Specifications of the equipment employed are presented in Table I. Reagents and Materials. All chemicals used were of reagent or analytical reagent grade. The phosphorus stock solution (1000 fig/mL) was prepared by dissolving potassium dihydrogen phosphate (Analar grade, Hopkin and Williams, Chadwell Heath, U.K.) in distilled water. Analyzed milk powder samples were kindly supplied by Cow and Gate Baby Foods, Trowbridge, Wiltshire, U.K. Procedure. Approximately 0.5 g of the milk powder to be analyzed was weighed accurately and mixed with 10 mL of glacial acetic acid. This mixture was then diluted to 100 mL with distilled 1492
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Table I. Instrumentation Plasma power supply IPC model 120-27; operating frequency: 27.12 MHz; power output: 0-2 kW continuously variable. Work coil: 1 1 / * turns 6mm 0.d. copper tubing, internal diameter of coil 26 mm. Spectrometer Hilger Monospek 1000: CzernyTurner scanning monochromator with plane grating (1200 lines mm-') blazed at 300 nm; reciprocal linear dispersion 0.8 nm mm-I. Optics Plasma imaged in 1:I ratio onto entrance slit with two fused silica lenses (7.5-cm focal length, 4-cm diameter). Readout Signal from EM1 6256 B photomultiplier tube displayed on Servoscribe chart recorder, model RE 511. Plasma torch Demountable fused silica torch with brass base. Coolant gas tubing: 18-mm i.d. Plasma gas tubing: 16-mm 0.d. Injector tubing: 6-mm o.d., 1.5-mm orifice diameter. Nebulizer Concentric glass nebulizer and spray chamber (Plasma-Therm Inc., Kresson, N.J.). Uptake rate: 1.3 mL min-I for water, 1.2 mL min-I for 10% v/v acetic acid; at argon flow rate 1.3 mL min-' . Efficiency for water, 3%. Efficiency for 10%v/v acetic acid, 4%. Table 11. Plasma Operating Conditions Net forward R F power Spectrometer slits Argon coolant gas flow rate Argon plasma gas flow rate Argon sample transport gas flow rate Viewing height
1200 w 20 p entrance and exit slits 11.0 L min-' 1 . 0 L min" 1.3 L min-I
25 mm above work coil
water to produce a stable solution. Sample solutions prepared in this way contained between 5 and 40 fig/mL phosphorus. These were analyzed by measurement of the phosphorus emission intensity at 213.62 nm produced on nebulization of the sample solutions into the plasma and comparison with a calibration graph constructed by measurement of the intensity at 213.62 nm produced on nebulization of aqueous phosphate standard solutions prepared in 10% acetic acid solution. The plasma operating conditions employed are shown in Table 11. These were chosen to optimize the signal-to-noise ratio observed at the phosphorus 213.62 nm line. RESULTS AND DISCUSSION Choice of Analytical Line for Phosphorus Determination. Although the principal resonance lines of the
I
Table 111. Determination of Phosphorus Content of Milk Powders
I
I
I
I
10
15
20
1
Phosphorus content, % Calcium By AOAC By Sample content, molybdate ICP-OES No. % methoda method RSD, % 1
2 3 4 5 6 7 a
0.36 0.72 0.78 0.73 0.7 5 0.93 0.46
0.22 0.42 0.15 0.49 0.17 0.64 0.42
0.21 0.44 0.17 0.50 0.17 0.62 0.42
2.2
-
-
2.0 1.4
Estimated precision, * 3%.
phosphorus atom all lie below 200 nm, there are a number of atomic emission lines in the range 200-300 nm which are of sufficient intensity to be analytically useful. In order to establish the most suitable of these lines for determination of phosphorus, the relative intensities and detection limita for the 213.62,214.91, 253.56, and 255.33 nm lines were measured; the 213.62 nm line exhibited the highest intensity and the lowest detection limit (0.1 pg mL-’) and was therefore selected for use in all further work. Effect of Calcium. The interference of phosphate in the determination of Ca is well known in flame atomic spectrometry and has been explained by the postulation that a relatively nonvolatile compound is formed between calcium and phosphorus in the flame (8). A number of studies of the effect of phosphate on calcium emission intensity in the ICP have been reported ( S I I ) . These have indicated the absence of any significant interference in the plasma tail flame. This might be expected as a result of the large amount of thermal energy available in the ICP source combined with the relatively long residence times of the particles in the axial channel of the plasma. The effect of the concomitant calcium ion on the intensity of phosphorus atom line emission observed in an early form of “tear drop” shaped ICP has been investigated by Kirkbright, Ward, and West (7); a 20% enhancement in the phosphorus emission intensity at 213.62 nm in the presence of a 50-fold excess of calcium was reported. As relatively high calcium levels are present in milk, it was decided to investigate the effect of calcium on the phosphorus determination with the source employed in this work. The results obtained at an observation height of 25 mm above the work coil demonstrated the absence of any significant interference in the presence of up to a 10-fold weight excess of calcium. A slight (4%) suppression was observed a t a 50-fold excess but this is not important for the purposes of the present work, as calcium concentrations of this order are not encountered in milk powder samples as is evident from Table 111. These results are in general agreement with the complementary observations (IO) of the effect of phosphate on calcium emission in the ICP source. Analysis of Milk Powders. The accuracy of the method was examined by determining the phosphorus content of seven milk powders which had been independently analyzed by the standard AOAC solution spectrophotometric method (7.095) after wet ashing. Initial attempts to analyze the samples after their dissolution in hot or cold distilled water gave rise to low results for the majority of the samples. This was attributed to incomplete dissolution of phosphorus-containing protein component.? in the milk powders. This problem was overcome by the use of acetic acid in the procedure employed to prepare the sample solutions. Addition of glacial acetic acid to the milk powders resulted in the formation of a fine precipitate
5
ACETIC ACID CONCENTRATION 1%)
Flgure 1. Effect of acetic acid on the emission intenslty at 213.62 nm of a 10 pg mL-’ phosphorus solution
which could then be easily taken up by dilution with distilled water to form a homogeneous solution which did not settle out on centrifuging. The presence of acetic acid in sample solutions was found to result in an enhancement of the phosphorus emission intensity at 213.62 nm as shown in Figure 1. This may be attributed in part to the effect of the changed physical properties of the solution (surface tension, density, and viscosity) on the nebulization efficiency as shown in Table I. Thus, in the determination of the phosphorus content of the milk samples, it was necessary to construct the calibration curve from aqueous phosphorus standards containing a concentration (10%) of acetic acid equivalent to that present in the sample solutions. The results obtained for the phosphorus determinations using the recommended procedure are shown in Table 111. These can be seen to be in good agreement with the results obtained by independent analysis. Precision. The precision of the complete method was estimated by carrying out replicate analyses on three of the milk samples (sample numbers 1,6, and 7 in Table 111). Four separate solutions were prepared for each of the three samples and four replicate analyses were then made on each of these solutions. Intensity measurements were obtained for sample solutions by wavelength scans through the 213.62 nm line and these were compared with the emission intensity observed for a standard phosphorus solution measured in a similar manner. The relative standard deviations in the results of the 16 analyses for each sample were calculated and are shown in Table 111. An overall value for the precision of the complete method was also obtained by computing the pooled relative standard deviation for the three series of analyses (48 samples); a relative standard deviation of 2% was obtained in this way. ACKNOWLEDGMENT The authors thank J. V. Stevens, Cow and Gate Baby Foods, Trowbridge, Wiltshire, U.K., for the supply of the analyzed milk samples. LITERATURE C I T E D (1) G. W. Dickinson and V. A. Fassel, Anal. Chem., 41, 1021 (1969). (2) V. A. Fassel and R. N. Kniseiey, Anal. Chem., 48, 1110A (1974). (3) P. W. J. M. Bournans and F. J. de Boer, Spectrochlm. Acta, Part 6 , 30, 309 (1975). (4) M. H. Abdaliah, R. Dlerniaszonek, J. Japosy, J. M. Mermet, J. Robin, and C. Trassy. Anal. Chim. Acta, 84, 271 (1976). (51 S. Greenfield and P. B. Smith. Anal. Chlm. Acta. 59. 341 (1972). i6j R. N. Knkley, V. A. Fassel, and C.C.Butler, Clin. Chem. ( W/nston-‘Sakm, N.C.), 19, 807 (1973).
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(7) G. F. Kirkbright, A. F. Ward, and T. S . West, Anal. Chim. Acta, 82, 241 (1971). (8) G.F. Kirkbright and M. Sargent, “Atomic Absorption and Fluorescence Spectroscopy”, Academic Press, London, 1974,p 516. (9) S. Greenfield, I. L. Jones, and C. T. Berry, AnalVst(London), 89, 713 (1964). (IO) G.F. Larson, V. A. Fassel, R. H. Scott, and R. N. Knlseley, Anal. Chem., 47,238 (1975).
(11) M. H.Abdallah, J. M. Mermet, and C. Trassy, Anal. Chim. Acta, 87, 329 (1976).
RECEIVED for review April 5, 1977. Accepted June 3, 1977. We thank the British Council for the award of a Postdoctoral Scholarship to one of us (L.N.O.).
Colorimetric Determination of Hippuric Acid in Urine and Liver Homogenate Shinji Ohmorl” and Mikiko Ikeda Faculty of Pharmaceutical Sciences, Okayama University, Okayama 700, Tsushima-Naka-I , Japan
Shohel Kira and Masana Ogata Department of Public Health, Okayama University Medical School, Okayama 700, Shikata 2-5, Japan
To a dried extract Containing hippuric acid (HA), 1.0 mL of acetic anhydride and 2.0 mL of 0.5% p-dlmethyiamlnobenzaldehyde (DAB) solution In pyridine were added, and the solution was kept at 40 O C for 1 h after thorough mlxing. The absorbance was then determlned at 458 nm agalnst a blank containing acetic anhydride, DAB, and pyridine. This method was in good agreement with Beer’s law within 1 to 100 pg and the mean f SD absorbance for 20 pg HA was 0.939 k 0.013 ( n = 5). The apparent molar absorptivity of 2.5 X 104/mol/cm, and the relative standard deviation of 1.4% was calculated.
The determination of hippuric acid (HA) in urine is of great significance, mainly for testing of liver function ( I ) , diagnosis (2-4), and estimation of the detoxication of alkylbenzenes and drugs (5-7). In this paper a convenient and reproducible method based on colorimetry is presented. About 40 papers dealing with methods of determination of HA have been published. These methods depend on column chromatography (8), extraction (9), colorimetry (10-13), gas chromatography (14-1 7)) thinlayer Chromatography (18), paper chromatography (19), fluorimetry (20), titration (21),and determination of radioactivity (22). The method most widely used at present is based on gas chromatography. Applying this method to the determination of HA in liver homogenates of rat and eel (instead of urine), we had difficulties with overlapping peaks. The method to be described here is applicable to urine as well as liver homogenates. Two colorimetric methods have been presented for the determination of HA. With the method of Umberger (10) which was later modified by Ogata, the color is produced in a mixture of the HA-containing sample with benzene-sulfonylchloride and pyridine. With the method reported by Gaffney et al. (19)and Ogata et al. (23),DAB is used. Gaffney e t al. employed paper chromatography. They detected the HA spot by spraying a 4% solution of DAB in acetic anhydride which contained a few crystals of sodium acetate with subsequent heating of the chromatogram at 130-150 “C for 1-2 min. The absorbance of the color was determined after elution of the spot. Ogata et al. improved this method. Their reaction 1494
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mixture additionally contained silica gel. The colorimetric procedure reported in this paper is based on the DAB method.
EXPERIMENTAL Chemicals and Instruments. Analytical grade chemicals from Wako Pure Chemical Industries Ltd (Osaka, Japan) were used. A Hitachi-139 Spectrophotometer was used for measuring the absorbance at 458 nm and a Hitachi-124 Spectrophotometer for recording the absorption spectra. Determination of HA. Extraction of HA from Urine. Urine (0.1 mL), 10 pL 6 N HC1, ca. 20 mg NaC1, and 1 mL ethyl acetate in a 10-mL test tube were mixed for 30 s with a Vortex mixer (Thermonics Inc., Japan). After 5 min, a 0.1-mL aliquot of the ethyl acetate layer was transferred to a 20-mL test tube and evaporated to dryness under reduced pressure. Extraction of HA from Liver Homogenates. Method A: for homogenates containing more than 20 pg HA per 0.5 mL. Rat liver (10 g) was homogenized in 90 mL of 1.15% KC1 in a Potter-Elvejhem Teflon pestle homogenizer and then centrifuged at 1000 X g for 10 min. To a 10-mL glass stoppered centrifuge tube, 0.5 mL of the supernatant and 20 pL 6 N HC1,20 gL glacial acetic acid, ca. 150 mg NaC1, and 2.5 mL ethyl acetate were added. The mixture was agitated with a Vortex mixer for 30 s and centrifuged at 1500 X g for 5 min. The ethyl acetate layer was dried over anhydrous sodium sulfate. An aliquot (0.5 mL) was then evaporated under reduced pressure. Method B: for homogenates containing less than 20 pg HA per 0.5 mL. To a 10-mL glass stoppered centrifuge tube 0.5 mL of the supernatant of the homogenate and 50 pL 6 N NaOH, ca. 150 mg NaC1, and 5 mL ethyl acetate were added. The mixture was agitated with a Vortex mixer. It was then centrifuged at 1500 x g for 10 min and the ethyl acetate layer was removed as thoroughly as possible. The aqueous layer was acidified with 0.1 mL 6 N HCl and extracted with 5 mL n-hexane by vortexing and centrifuging. After the hexane layer was removed as thoroughly as possible, the aqueous layer was extracted with 5 mL ethyl acetate by vortexing and centrifuging. The extract was dried with anhydrous sodium sulfate and an aliquot (3.5 mL) was evaporated under reduced pressure. Development of the Color and Its Measurement. To the dried HA-containing residue, 1.0 mL of acetic anhydride and 2.0 mL of 0.5% DAB solution in pyridine were added in turn. After thorough mixing, the solution was kept at 40 O C for 1 h. The absorbance was then determined at 458 nm against a blank containing acetic anhydride, DAB, and pyridine.
