pectrophoto etric Determination of Phosphorus R. K. Skogerboe, Ann S. Gravatt, and G.)-I. Morrison Department of Chemistry, Cornell Unicersity, Itliaca, N . Y . Flame conditions which permit the excitation of the atomic spectrum of phosphorus are reported. The analytical utilities of the atomic spectra and the phoshorus monoxide bands are compared for different flame types and detection limits are given. Interference effects are evaluated and a simple batch ion exchange separation is given for the elimination of these effects. The method has been applied to the analysis of representative biological materials and the results are shown to compare favorably with colorimetric results.
FLAME SPECTROPHOTOMETRY has been used with great success for the determination of many trace elements but the application of this technique t o the determination of phosphorus has been limited by the inability to obtain adequate sensitivity. Although Gilbert ( I ) has suggested the possibility of using the phosphorus monoxide band a t 246.4 mp, no applications have appeared to date. Brite ( 2 ) and, more recently Davis, et al. (3) used an oxyhydrogen flame with aqueous-organic solutions to excite the phosphorus oxide continuum centered a t 540.8 mp and determine the relatively high phosphorus content of organic and inorganic phosphorus compounds. Flame excitation of the atomic spectra of this element has not been reported, presumably because the atomic excitation potentials and the stabilities of the phosphorus molecular species involved are high in comparison to those of many other elements. In the present investigation excitation of the atomic spectra has been accomplished and the conditions which maximize the atomic emission have been determined. The analytical potentialities of both atomic and molecular spectra for the oxyacetylene, oxyhydrogen, and air-hydrogen flames were also evaluated. Interferences in the trace determination of phosphorus by the flame method have been investigated and a simple batch ion exchange separation procedure was developed t o eliminate these problems. The method has been applied to a number of representative biological materials. The study indicates that phosphorus can be rapidly and accurately determined by flame spectrophotometry down to a concentration level of 5 pgiml. EXPERIMENTAL
Apparatus. The instrumentation used and the operating parameters which remained fixed throughout the study are listed in Table I. Reagents. Analytical reagent grade phosphoric acid and metal chlorides were used to prepare phosphorus and metal glycerol was solutions in absolute ethanol; 0.5 volume added t o prevent precipitation of metal phosphates where required. Dowex 50W-X8 ion exchange resin in the hydrogen form was utilized for the separations.
(1) P. T. Gilbert, Jr., “Xth Colloquium Spectroscopicum Internationale,” Spartan Books, Washington, 1963, p. 171. (2) D. W. Brite, ANAL.CHEM., 27, 1815 (1955). (3) A. Davis, F. J. Dinon, E. J. Lobbett, J. D. Chazin, and L. E. Tufts, Ibid.,36, 1006 (1964). 1682
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Table I. Experimental Apparatus and Conditions Excitation system Burner Beckman $4020, oxyhydrogen; $4030 oxyacetylene Burner mount Custom built rack and pinion Gas regulation Pressure Custom built Beckman type regulators; 10-psi hydrogen pressure, 20-psi oxygen pressure Flowmeters 1 rotameters; Brooks Instrument Co., Model #6-1110-24-MFB Control valves ‘/&ch needle valves at rotameter outlet; Hoke Inc., 280 series Sample delivery Continuously variable infusion pump ; Harvard Apparatus Co., Model #600900VDC
Dispersing system Monochromator Grating Slits External optics Readout system Power supply and amplifier Photomultiplier Recorder
Jarrell-Ash $82000 scanning Ebert, 0.5meter focal length 1200 line/mm, blazed for 3000 A Unilateral adjustable straight slits 10-cm spherical quartz condensing lens focused on the entrance slit to sample a 5-mm-high segment of the flame ORNL design ( 4 ) EM1 6255B, 13 stage, S-13 response, operated at 1900 volts Leeds and Northrup, Model G modified for continuously adjustable 1-20-mV range and =jO-mV zero suppression; IO-mV span used
RESULTS AND DISCUSSION
It has been shown that the optimization of the controlling parameters of the flame is of crucial importance in order lo achieve lower limits of detection (5). Temperature, geometry, droplet-size distribution, sample feed rate, and the populations of species which may participate in dissociation and/or excitation producing reactions are characteristics of the flame which must be regulated at optimal lebels. The selection of these levels for a particular solvent can be best accomplished by varying four controlling parameters : the fuel and oxygen flow rates, the flame region viewed by the spectrometer, and the sample feed rate. The use of a n infusion pump to regulate sample delivery independent of the oxygen flow rate is particularly advantageous because of the added control and the increase in both experimental range and flexibility as compared t o normal burner aspiration (5). Figure 1 indicates the effects of each of the controlling variables on the emission intensity of the atomic line and the ultraviolet PO band a t the optimum levels for the other variables. The atomic spectra were observable only in the oxyacetylene flame supported by a n organic solvent. Several factors should (4) M. T. Kelley, D. J. Fisher, and H. C. Jones, ANAL.CHEM., 31, 178 (1959). (5) R. K. Skogerboe, Ann T.Heybey, and 6. H. Morrison, ANAL. CHEM.,38, 1821 (1966).
a CD
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1
2
3
4
5
Sample Feed Rote, ml/rnin
a
x
(b)
>
m in
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in N
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Figure 1. Effects of flame variables on emission intensities
be particularly noted with regard to the atomic emission. Maximum atomic emission is highly localized in the interconal region of the flame (Figure l b ) and was not observable at the normal burner aspiration rates (1.5 m1;'minute). In fact, unusually low sample feed rates were required to maximize the emission intensity (Figure l a ) . In contrast to other elements with a tendency to form stable oxides in the flame, phosphorus does not require a particularly fuel-rich flame (Figure IC and Id). Rather, the maximum emission intensity occurred at an oxygen-to-fuel ratio of 1.6 which approaches the stoichiometric ratio. It is possible that a stoichiometric flame is actually optimum but extreme instability of the flame at high oxygen flow rates precluded the use of rates above 5.2 liters/ minute (Figure IC). These observations: the low sample feed rate, interconal emission, and the nearly stoichiometric flame are all consistent with higher temperatures and consequently are indicative (although certainly not conclusively) that the atomic excitation of phosphorus is primarily of thermal origin. Figure 2 presents recordings of the 253.6- and 255.5-ml doublets of phosphorus. The detection capability at these wavelengths is limited by two factors: the small monochromator slit width necessitated by the high background of the fuel-rich flame, and interfering emission peaks. The latter are present at wavelengths coincident with the atomic doublets and are attributable to molecular emissions of carbon species, The effect of the solvent on the atomic emission was checked with isopropanol, isobutanol, and 4-methyl-2-pentanone as well as ethanol. The optimum flame conditions were essentially the same for all solvents and the detection capabilities agreed within 10% so ethanol was chosen for all subsequent
L
Figure 2. Atomic spectrum of phosphorus (900 Pglml)
studies, The optimum analysis conditions determined are presented in Table 11. The ultraviolet phosphorus monoxide bands are readily excited in the three flame types listed in Table I1 when an organic solvent is used. Again, however, the high background of oxygen-supported flames requires the use of reduced slit widths with concomitant losses in detection sensitivity. The very low background of the air-hydrogen flame permits the use of a large band pass with a resultant gain in detection capability. The optimization data presented in Figure 1 for the excitation of this species in the air-hydrogen flame also indicates the advantage of a sample delivery system external to the burner. In this instance, the optimum sample feed rate is considerably greater than the rate possible when the burner performs its own aspiration function. The broad, somewhat nonspecific, continuum at 540.8 mp originating frgm unvaporized PO ( 2 , 3) was observed with flames using hydrogen as fuel. The use of acetylene flames was precluded by background limitations in this spectral region. Detection Limits. The general method used to determine the detection limits given in Table I1 has been presented (5). Because band spectra were used here and because emitting carbon species interfere with the atomic lines, intensities were
Table 11. Optimum Excitation Conditions
Oxidant Wavelength, mP 253.6 246.4 540.8
Fuel
Flame type
flow rate, l/m
Oxyacetylene Oxyacetylene Oxyhydrogen Air-hydrogen Oxyhydrogen Air-hydrogen
3.2 3.2 3.2 10.0 7.8 8.4
flow rate,
l/m
Flame region, mm above burner tip
Sample feed rate, ml/m
Slit width, p
Detection limit, pg/ml
5.2 5.5 4.5 4.6 4.5 4.1
18 21 21 32 19 21
0.4 0.4 3.7 3.7 1.2 1.6
6 10 10 140 30 30
400 900 400 5 100 500
VOL. 39, NO. 13, NOVEMBER 1967
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Table 111. Comparison of Flame and Colorimetric Analysis Sample type Liver Cherry leaves Bone
1 . 5 =t0 . 3 0.162 =t0.005 9.8 i1.5
I
I
I
20
30
40
50
1.3 i 0 . 1 0. I68 tt 0.006 9.7 i 1 . 0
(6) R.Herrmann and C . T.J. Alkemade, “Chemical Analysis By Flame Photometry,” Interscience, New York, 1963, p. 304. e
I
Weight oer cent ohosahorus Flame Colorimetry
measured without scanning. The solvent was used to define the baseline in each instance. Concentrations required to produce a signal twice the standard deviation of the blank signal were taken as detection limits. On the basis of the data in Table I1 the ultraviolet band, air-hydrogen flame is the most obvious analytical choice. Interferences. It is generally accepted ( 6 ) that the depressant effect of phosphate on calcium (and vice versa) can be attributed to the formation of nonvolatile calcium-phosphorus species in the flame. On the basis of this mechanism interferences from other elements with a tendency for the same type of compound formation were anticipated. Thus the chemical interference effects of Ca, Ba, Ag, and Fe were investigated to obtain a representative indication, Solutions containing equimolar amounts of P and the respective metals were compared with a similar solution containing only phosphorus to ascertain the interference effects. In the airhydrogen flame Ca, Ba, and Ag depress the 246.4-mp monoxide band intensity by 90, 32, and 6%, respectively, while Fe enhances the intensity 23 %. Although only the most serious interfering elements have been checked, the results are indicative of potential problems and suggest the necessity of separation before analysis. This is particularly important in the case of biological materials which normally have high concentrations of calcium and iron among other potential interferents. With regard to the 540.8-mp band head, Davis and associates (3) list a number of interferences. An examination of the flame spectrograms given by Herrmann and Alkemade ( 6 ) indicates that most of the listed interferences are caused by emission in the same spectral region. In fact, Ca, Ba, Na, K, several transition metals, and most rare earth elements, (totaling nearly 30 elements) can be expected to cause spectral interference problems at this wavelength depending on the concentrations, the flame type, the flame conditions, and the solvent. Most of these elements exhibit oxide and hydroxide band emission in this region, the intensity of emission being generally most prominent in hydrogen flames. For this reason, the analytical use of the 540.8-mp band is perhaps unrealistic except for limited applications such as those mentioned (2, 3). On the other hand, the atomic lines and the ultraviolet band are relatively free of spectral interference from other species which may be present in samples. With the instrumentation and conditions utilized, the most sensitive atomic line at 253.6 mk is subject to interference only from iron. Depending upon the flame conditions cobalt, iron, arsenic, antimony, tin, lead, and nitrous oxide are potential spectral interferents for the ultraviolet band head (6). This, in fact, is the reason for the iron enhancement effect reported above. In view of the interference problems, particularly those of a
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‘0
10
60
T i m e , min
Figure 3. Batch separation times for removal of interfering elements chemical nature, separation of the phosphorus becomes a practical necessity. Separation. Because phosphorus normally exists in anionic form in solution, its separation can be readily accomplished utilizing a cation exchange resin. To simplify the operative problems a batch ion exchange separation was investigated, The solutions used to obtain the interference data given above were mechanically shaken with an excess of resin and the intensities of the flame emission for the respective metals were used as criteria for completeness of separation. Figure 3 presents such data for the separation of Ca, Fe, and Ba. After 30 minutes of shaking the metal, concentrations were reduced to a negligible level and their respective effects on the phosphorus emission were completely eliminated. Applications. To verify the applicability of the combined separation-flame spectrophotometric technique to the analysis of biological materials a representative variety of the latter were analyzed and the results compared with molybdovanado-phosphoric acid spectrophotometric analyses (7). Cherry leaves, liver, and bone samples were oven-dried at 60’ C and 1-mm pressure, followed by grinding and blending. The grinding and blending was effected easily in the case of the cherry leaves, but some difficulty was experienced with liver and bone because of their hydroscopic nature. One-gram samples of the dried materials were wet-digested in nitricperchloric acid media, evaporated to near-dryness, redissolved in 2 to 3 ml of 2Nnitric acid, and diluted with absolute ethanol in ethanol. The to produce a final solution 90 vol solutions were subsequently shaken with 10 grams of the dry resin for the specified time except for the bone sample which required 60 minutes t o remove the high calcium content. After filtering off the resin, the analyses were run using the 246.4-mp band head, the air-hydrogen flame adjusted to the conditions specified in Table 11. Phosphoric acid standards which had been equilibrated with the resin were similarly run. Comparative results by the flame and colorimetric methods expressed as weight per cent referred t o the dry weight are presented in Table 111. All results agree within the limits of error (expressed as the standard deviation of four complete analyses of each sample) of the two methods. The precision estimates reflect the overall error including sampling, chemical processing, and measurement errors. The relative standard deviation of the measurement step for all three samples was (7) R.E. Kitson and M. G. Mellon, ANAL.CHEM., 16,379 (1944).
2 to 3 %. The poorer overall precision for the liver and bone samples reflects the less effective homogenization of the material for analytical sampling. CONCLUSIONS
allow analyses for these elements without attendant chemical interferences such as the phosphate and sulfate effect on calcium and magnesium. The ability to excite the atomic spectra suggests that other fuel-rich, higher temperature media such as the nitrous oxide-acetylene flame might also be used.
The flame determination of phosphorus in most biological materials can be reliably and rapidly accomplished by applying the simple separation technique used above. Subsequent recovery of the metallic species by elution from the resin would
RECEIVED for review May 15, 1967. Accepted August 24, 1967. Research supported by the National Science Foundation.
Determination of Nitrite Ion Using the Reaction with p -Nitroaniline rad Azulene E. E. Garcia Nuclear Technology Department, General Electric Co., Pleasanton, Calif. A sensitive, rapid method for the determination of nitrite ion i s described. Nitrite is reacted with p-nitroaniline to form a diazonium ion, which is then coupled with azulene to form a purplish pink azo dye. The absorbance of the dye is measured at 515 mp. Diazotization and coupling are very fast and no controls of pH and temperature are necessary. The sensitivity of the method (expressed as the amount of nitrite corresponding to an absorbance of 0.010 at 515 mp in a 1-cm cell) is 0.22 pg of nitrite. The molar absorptivity is 5.2 X l o 4 mole-1 cm-1 liter. The precision i s also satisfactory; the relative standard deviation at the 10-119 level is &1.6%. The procedure’s simp!icity has permitted its use in remote analyses of highly radioactive solutions. Iron(lII) is the only ion tested that interfered seriously. Large amounts of nitrate and nitric acid do not interfere.
NUMEROUS PROCEDURES are available for the determination of the nitrite ion in solution. Good reviews can be found in the literature (1-3). The spectrophotometric procedures normally recommended involve the diazotization of an aromatic amine and the coupling of the diazo compound with a naphthalene derivative to form a highly colored, relatively stable, azo dye.
manipulation in a hot-cell, and classical spectrophotometric procedures were critically evaluated. Most of them offer excellent sensitivity and specificity, but quite often require close control of pH and temperature during the diazotization step, as well as a relatively long coupling time. Because of the severe limitations posed by remote manipulation, and the unknown, but possible, effects of very high radiation levels on samples and reagents, simpler procedures were desirable, and a review of the more recent literature was made. Sawicki and his coworkers ( 4 ) at the U. S. Public Health Service compared the relative merits of 52 procedures for nitrite ion determination, and included outlines of some 30 methods not reported before. Based on considerations of simplicity, procedural time, color stability, adherance to Beer’s law, sensitivity, and availability of reagents these procedures were rated, and one in which p-nitroaniline and azulene were used was selected for further evaluation. The reaction sequence in the procedure involves two steps. In the first, p-nitroaniline reacts with nitrite to form the diazonium ion. In the second step, the diazonium ion is coupled with azulene to form a purplish pink azo dye. The reactions can be represented as follows :
N
G Recently, it was necessary in this laboratory to determine traces of nitrite in highly radioactive solutions by remote (1) D. F. Boltz, “Colorimetric Determination of Non-Metals,” Interscience, New York, 1958, p. 130. ( 2 ) I. M. Kolthoff and P. J. Elving, ‘Treatise on Analytical Chemistry,” Part 11, Vol. 5, Interscience, New York, 1963, p. 275. (3) E. Sawicki, J. Pfaff, and T. W. Stanley, Rec. Unic.Ind. Santander, (Columbia),5 , 337 (1961).
N02 The addition of the azo compound in position 1 is supported experimentally and theoretically (5). The color of the (4) E. Sawicki, T. W. Stanley, J. Pfaff, and A. D’Amico, Talanta, 10,641 (1963). (5) D. Ginsburg, “Non-Benzenoid Aromatic Compounds,” Inter.
science, New York, 1959, pp. 171-337. VOL. 39, NO. 13, NOVEMBER 1967
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