fractory metals and alloys are only incompletely attacked by the HF-dichromate combination. The function of the phosphoric acid is not fully understood. In the case of tungsten and molybdenum, the formation of phosphotungstic or phosphomolybdic acid can be conjectured as aiding the dissolution of the sample. Ferric salts may also be complexed by phosphoric acid. No mechanism is apparent to account for the role of phosphoric acid in dissolving nickel, cobalt, niobium, or tantalum. It was also found that phosphoric acid prevents the formation of CrF3 which is otherwise only slightly soluble in hydrofluoric acid. Other Acid Combinations. In the case of ch-omium base alloys, it was noted that a more rapid decomposition of the sample can be achieved if part of the hydrofluoric acid is replaced by 2.5M hydrochloric acid. Effect of Nitrate Ion in Proposed Procedure. Because in the proposed method the nitrate ion must compete with a large excess of dichromate ions, it was conjectured that nitrates would be less harmful than in methods using unstable oxidants. See Table I. The data in Table I confirm that in the proposed procedure only a fraction of the nitrate is reduced.
RESULTS
The suggested method was applied to the determination of nitrogen in the four MAB samples (3), four Air Force samples (4), and several other samples for which data on nitrogen were available. In some instances, where nitrogen data were not available, both the dichromate and the bromate methods were used. The results presented in Table I1 indicate that the proposed method is suited for the determination of trace amounts of nitrogen in a variety of refractory metals and alloys. Steel samples were included only to prove the applicability of the methods. Most steel samples can be dissolved in hydrochloric acid without the addition of an oxidant. Although not within the stated scope of this paper, it should be mentioned that the proposed method is equally applicable to samples containing large amounts of nitrogen. Most nitrides are readily attacked by the HF-H3PO4-K2Cr2O7combination. It was found that the method is particularly suited for the determination of the N/Uratio in uranium nitrides containing various metal additives. RECEIVED for review October 20, 1967. Accepted November 29, 1967. Work supported in part by Air Force Contract NO. F3361-67-C-1524.
Atomic Fluorescence Spectroscopy of Silver Using a High-Intensity Hollow Cathode Lamp as Source T. S . West and X . K . Williams Chemistry Department, Imperial College, London S . W . 7 , England The fluorescence of silver atoms in air-propane and air-acetylene flames is described. With a high intensity hollow cathode lamp as source, resonance fluorescence at 3280.7 and 3382.9 A is obtained, and none at any of the other lines emitted strongly by the source. The effects of variables such as primary and secondary cathode currents, amplifier-gain, scaleexpansion, distance between source, flame and detector, etc., are studied. Silver may be determined easily in the range 0.01 to 10 ppm in aqueous solution and, following extraction as di-n-butylammonium silver salicylate, down to 0.5 ng per ml (5 X 10-4 ppm) at 3280.7 A. Detection limits of 1.7 X ppm and 4X ppm were found for aqueous solutions without and with extraction, respectively. No interference was found from thousandfold excesses of a range of foreign ions examined, but elements which form refractory oxides, e.g. aluminum, reduce the sensitivity. No problems were encountered from scatter of excitation source radiation. Observations are also made on the use of the high intensity hollow cathode lamp as source in atomic absorption measurements of silver in air-propane, air-acetylene, and nitrous oxide-acetylene flames. An oxidizing air-propane flame is best for both atomic absorption and atomic fluorescence measurements.
of the incident and emergent light signals. As a result, the sensitivity of atomic fluorescence spectroscopy (AFS) may be improved within reasonable limits by increase of amplifier gain and source intensity whereas atomic absorption spectroscopy (AAS) remains unaffected by these factors. Up to the present time AFS has been effected with several excitation sources. The use of continuous sources such as the xenon arc lamp (1, 2) has demonstrated that a single (continuous) source may be used to excite atomic fluorescence for many elements-e.g., Ag, Au, Bi, Cd, Cu, Co, Fe, Hg, Mg, Mn, Pb, TI, and Zn. The detection limits obtained for the above elements with the continuous source is comparable to that obtainable by AAS. The fact that only one source is required instead of one for each element as in conventional AAS is also a worthwhile practical consideration. Spectral discharge lamps for individual elements have the advantage of supplying the energy of excitation chiefly in the required atomic lines instead of dispersing it over the entire spectrum. consequently, the sensitivity of AFS using spectral discharge lamps is considerably higher than that obtained using continua. Elements determined in this way, (2-5), include Cd, Hg, T1, and
DETERMINATION OF several elements by excitation of their atomic fluorescence in flame media has been described recently. The technique makes more effective use of large ground-state populations of neutral atoms than does atomic absorption spectroscopy, by virtue of the linear dependence of the fluorescence signal upon the intensity of the source as well as the number of ground state atoms. With absorbance measurements, the signal depends upon the logarithmic ratio
(1) C. Veillon, J. M. Mansfield, M. L. Parsons, and J. D. Winefordner, ANAL.CHEM., 38, 204 (1966). (2) R. M. Dagnall, K. C. Thompson, and T. S. West, Ana/. Chirn. Acta, 36,269 (1966). (3) J. D. Winefordner and R. A. Staab, ANAL.CHEM.,36, 165 (1964). (4) R. M. Dagnall, T. S. West, and P. Young, Tuluntu, 13, 803 (1966). (5) J. M. Mansfield, C. Veillon, and J. D. Winefordner, ANAL. 37, 1049 (1965). CHEM., VOL 40, NO. 2, FEBRUARY 1968
335
Zn. With microwave-excited electrodeless discharge tubes as sources for individual elements, AFS methods have been described similarly for As, Bi, Ga, Ge, Hg, In, Sb, Se, Te and Tl(5-8). In the present paper we report the atomic fluorescence spectra of silver using a high intensity hollow cathode lamp of the type described by Sullivan and Walsh (9) to excite fluorescence and make comparison with the atomic absorption of silver using exactly the same equipment and source, but with a longer pathlength flame, and various flame media. EXPERIMENTAL Apparatus. A Techtron AA4 atomic absorption spectro-
photometer fitted with an Evans Electroselenium Limited flame emission burner head and A.S.L. high intensity silver hollow cathode lamp was used. The lamp and detector were modulated at 285 cps. Instrumental Settings. The instrumental settings were: primary lamp currcnt, 20 mA; booster current, 400 mA; wavelength 3280.7 A; slit, 300 p ; air-pressure, 15 psi; and propane pressure, just sufficient to produce a stable flame. In spraying the isobutyl methyl ketone extracts, the propane pressure was further reduced until a lean non-turbulent flame was obtained. (Care is necessary that the flame does not extinguish between spraying different organic extracts.) Reagents. A stock silver solution was prepared by dissolving 0.9128 gram of AgN03 in distilled water and diluting to 100 ml (5798 ppm of Ag). More dilute solutions were prepared as required. Solutions containing less than 10 ppm Ag should only be prepared immediately before use because of the marked tendency of silver to be adsorbed on glassware (3). A hexone extraction agent was prepared by dissolving 3.2 grams of salicylic acid and 20 ml of di-n-butylamine in isobutyl methyl ketone and diluting to 1 liter with the same solvent. Calibration Curves. Switch on the AA4 units with the lamp in the warm-up position and allow to stand for about an hour. Peak th? monochromator on the signal from the lamp at 3280 A. Situate the lamp at right angles to the optical bar of the instrument and, with the focusing lens and saddle removed, move the burner as close as possible to the slit, and place the lamp end-window about 1 inch distant from the flame. Spray a silver solution into the flame, adjusting the slit-width and gain controls so that a scale reading of about 50% is obtained. Adjust the height and position of the burner head and lamp so that the maximum fluorescence signal is obtained. Concentrations in the range 0.01-1 ppm Ag were determined using scale expansion and a calibration curve was constructed in the usual way. For the range 0.5-10 ppm Ag, a reduced amplifier gain setting without scale expansion was used. For the range 0.0005-0.03 ppm, a solvent extraction process was used as follows: Protect a 2-liter separating funnel with a protective coating of the silicone Repelcote (Hopkin and Williams Ltd.) and take 10-ml aliquots of solutions containing 0.05 to 3.0 ppm Ag and dilute to 1 liter in the funnel. Add 5 ml of 20% sodium acetate solution, 5 ml of 5 M sodium nitrate, and 50 ml of the hexone extracting agent. Shake the funnel by continuous inversion for 1 minute and chill thoroughly in ice water for about 10 minutes to obtain clear phases. Discard the lower aqueous layer and transfer the organic phase (6) R. M. Dagnall, K . C . Thompson, and T. S. West, Tu/arzta, 14, 551 (1967). (7) Ibid., p. 557. ( 8 ) Ibid., p. 1151. (9) J. V. Sullivan and A. Walsh, Spectrochim. Acta., 21, 721 (1965).
336
ANALYTICAL CHEMISTRY
(about 40 ml) to the 50-ml volumetric flask. Wash the funnel with 5 ml of hexone and transfer to the flask. Make the volume of the contents of the flask up to 50 ml exactly with hexone. Proceed as above with scale expansion ( X 5), full gain, and maximum slit width (300 p) for the range 0.0005-0.01 ppm Ag, or without scale expansion, but with maximum gain and slit width, for the full 0.0005-0.03 ppm range, Spectral Characteristip of Atomic Silver. Both the 3280.7 A line and the 3382.9 A line have been used in atomic absorption spectroscopy. Robinson (10) quotes detection limits of 0.1 and 0.15 ppm, respectively, for absorption at these wavelengths. In the only reported comparison of these two lines by atomic fluorescence spectroscopy Dtgnall, Thompson, and West (2) observed that the 3382.9 A line !ppeared to be about 40% more sensitive than that at 3280.7 A. However, a 150 W ac high-pressure xenon arc lamp was used for excitation and the spectral emission profile of the lamp shows an increase of intensity with wavelength in this region as does the response of the EM1 9605B photomultiplier used in the S.P. 900A flame-spectrometer. Thus, though the atomic fluorescence was stronger in practice at the longer wavelength, this was probably due to a combination of instrumental responses qther than to a fundamentally greater sensitivity at 3382.9 A. The absorbance data, which are virtually independent of such instrumental effects, should give a better index of sensitivity. Intensity of Source. In the high intensity hollow cathode lamp, the photoemission is controlled by the (primary) cathode current and by the (secondary or booster) current flowing between the two auxiliary electrodes which discharge across the cloud of cold atomic vapor at the exit of the cathote. It was found that the intensity of fluorescence at 3281 A for a given solution of silver increased with both primary and secondary lamp currents. The highest sensitivity is obtained by running the source at the maximum operating currents stipulated by the manufacturers of the lamp-viz., 20 mA for the primary current and 400 mA for the secondary current. It should, however, be noted that practically no fluorescence signal can be obtained even with 500 mA secondary current and low primary cathode currents (e.g., 1-3 mA) because insufficient atomic vapor is produced. Positioning of Lamp. The lamp was situated at right angles to the plane of the flame and the entrance slit of the monochromator as in all previously reported studies of atomic fluorescence spectroscopy. In some instances where high concentrations of silver were being nebulized into the flame, the lamp was positioned to subtend an acute angle with this plane to see if frontal illumination of the flame might minimize the effects of re-absorption of fluorescence (generated in the hot regions) by cold atomic vapor in the outer sheath of the premixed flame. This, however, proved to be of no benefit. Because of the inverse square law, the most sensitive results were obtained by placing the end window of the hollow cathode as close as possible (about 1 inch) to the flame in the right-angled arrangement. Even so, the hollow cathode itself is about 41/2 inches farther away from the flame than the window and better sensitivity would obviously be obtained by a lamp designed to bring the cathode much closer to the window. The introduction of focusing lenses between the lamp and the flame and between the latter and the monochromator slit was not found to be beneficial, but the off-axis ellipsoid mirror arrangement suggested elsewhere might be advantageous (11). Choice of Flame and Flame Characteristics. The cylindrical Meker-type emission burner head supplied with the instru(10) J. W. Robinson, “Atomic Absorption Spectroscopy,” Marcel
Dekker, New York, 1966, p. 1489. (11) T. S. West, Chem. Ind. (London), 1966, 1005.
Table I. Detection Limits. for Atomic Fluorescence of Silver at 3280.7 A in Air-Propane and Air-Acetylene Flames Distance of burner from Scale Detection limits, PPm Fuel gas slit, inches expansion at 20 mAb at 10 mAb Propane 3.5 None 0.024 ... Propane 3.5 x5 0.004 0.009 Propane =I None 0.008 0.030 Propane =l x5 0.0017 0.006 Acetylene 3.5 None 0.046 * * . Acetylene 3.5 x5 0.010 ... Acetylene =I None 0.014 ... Acetylene =l x5 0.0037 ... Taken as ppm Ag+ equivalent to 1 % scale deflection at a signal : noise ratio of 2 : l . b Primary cathode currents. Note: Secondary cathode current = 400 mA; gain 20; slit 300 p ; air pressure 15 psi; lamp 1 inch from flame.
.a 9
m m
L
-
.a Q
N
11 to
10
Instrument gain
Figure 1. Emission from high intensity silver hollow cathode lamp, uncorrected for detector response Lamp currents 20/400 mA, slit 25 p ment was replaced by an Evans Electroselenium flame photometer emission head for air-propane because it was found to be more suitable than that supplied by the manufacturer. The air-propane head has two rows of five holes each, and it was positioned so that the rows were parallel to the optical axis of the monochromator. A Techtron emission-head was used for air-acetylene. In both instances, the best fluorescence signal was obtained when the radiation from the hollow cathode lamp was directed at the center of the flame in a position just above the primary cones of the burning gases. The height of the burner head was adjusted so that the primary cones were just below the horizontal plane of the bottom of the monochromator entrance-slit. The results shown in Table I indicate the superiority of the airpropane flame and show how the best sensitivity is obtained
with use of scale expansion and with the flame close up to the monochromator slit. The most suitable flame conditions were found to be obtained with a strongly oxidizing fuellean propane flame. Amplifier-Gain and Slit-Width. As a silver-specific source was used in these studies, it was possible to open the slit width to its maximum value of 300 p without adverse effect on selectivity or stability of the fluorescence signal. It is therefore best to reduce sensitivity where necessary by decreasing the amplifier gain rather than by decreasing the slit width. Considerable noise may be introduced when scale expansion is used. Choice of Atomic Line. Eight lines emitted by the source were studied. In order of increasing wavelength, these were the silver lines at 2061.2,a2069.8, 3280.7, 3382.9, 4055.3, 4210.9, 5209.b, and 5465.5 A. The resonance lines at 2061.2 and 2069.8 A were very weak indeed and as expected they showed no fluorescence in the flame. It is highly improbable that these ljnes enrich the fluorescence emission at 3280.7 and 3382.9 A, respectively, by step-wise fluorescence following radiationless transitions from the 6p 2POl~/, and 6p 2P01,2 to the 5p *POl~/,and 5p 2P01/, states. Figure 1 shows a scan of the other six lines at varying amplifier gains as emitted by the source and viewed by the R 213 photomultiplier following dispersion by the monochromator. It will be s e p that the two other resonance lines at 3280.7 and 3382.9 A are the most strongly $mitted. The lines at 4055.3, 4210.9, 5209.1, and 5465.5 A are relatively weak. The lamp emissions should of course be corrected for the variation of detector response at different wavelengths. We have calculated correction factors from data supplied by the lamp manufacturers and list corrected and uncorrected data inoTable 11. Only the resonance lines at 3280.7 and 3382.9 A could be detected in fluorescence from the flame.
Table 11. Relative Sensitivities of Silver Lines Emitted by Source and Fluorescedo in Air-Propane Flame Rel. sensitivity, Detectorb Rel. sensitivity, uncorrected response, corrected Atomic line, A Transition 2061.2 2069.8 3280.7 3382.9 4055.3 4210.9 5209.1 5465.5
Bracketed terms represent relative intensity (3280.7 A b Calculated from manufacturers' data.
0.04 0.01
...
...
lo00 (1OOo) 600 (600)
3 5 26 28 =
... *.. ...
...
44.2 45.0 63.5 73.5
98.0 99.75 85.5
78.0
0.058 0.014
.,, ...
1OOo (looo)
520 (500) 2 ... 3 ... 19 ... 23 ...
1OOO) in fluorescence.
VOL 40, NO, 2, FEBRUARY 1968
337
Table 111. Sensitivity of Atomic Absorption of Silver at 3280.7 A in Various Flame Media (Aqueous Solution)
Cathode current, mA Primary 20 10 2 2 10
'
Secondary 400 400 400 0 0
1
Detection limits (1 Airpropane" 0.23 0.2 0.16 0.17 0.26
Airacetylenes
0.37 0.25 0.197 0.203 0.580
Abs) pprn
N20acetyleneb 1.75 1.36 1.16 1.16 2.03
flame Identical nebulization pressures. 5-cmflame N.B. Utilizing the X5 scale expansion, these detection limits can be reduced 5-fold-i.e., propane at 2/400 mA = 0.03 ppm detection limit.
Figure 2 shows a scan of the fluorescence signals, and the uncorrected and corrected relative sensitivities for these lines are shown in Table I1 also. It is interestingotonote that the uncorrected ratios for the 3280.7 and 3382 A lines are 1 :0.6 in both source emission and flame-fluorescence and 1 :OS2 and 1 :OS, respectively, following correction. This presents good evidence for supposing that there is no stepwise fluorescence contribution to the resonance fluorescence of these lines and also that the quantum efficiency of the fluorescence process for both lines is identical. The relative sensitivities of the 3280.7 and 3382.9 A lines were determined from a practical standpoint by measuring the concentrations of silver required to produce a 1% transmission signal, with the instrumental settings (gain, slit-width, etc.) set well below their optimal values but with maximum lamp currents. This yielded vplues of 0.14 ppm and 0.20 ppm for the 3280.7 and 3382.9 A lines, respectively, revealing that the former line is about 30% more sensitive than the latter. It is interesting to note that this agrees extremely well with atomic absorption sensitivity data reported for these two lines by Robinson (10). In previous atomic fluorescence measurements done in this laboratory using a xenon arc lamp as source (2), this order of sensitivity was reversed. This we attribute to the sharp decline in !mission from a xenon arc source in the range 3500-3400 A as shown diagramatically in another communication from this laboratory (12). Range of Determination of Silver by AFS. The lowest concentration of silver determined in these studies was 0.01 pprn in aqueous solution using maximum amplifier gain and lamp currents. The maximum concentration of silver that could be determined while retaining a linear dependence of fluorescence signal to concentration was 10 pprn. Above 50 ppm, the fluorescence signal was actually found to decrease with increase of concentration. This point was also marked by a sudden onset of noise even at the low amplifier gain used for such measurements. This was initially thought to be due to source scatter, but experiments with as much as 5000 ppm of sodium chloride aspirating through the flame showed no measurable scatter signal. Apparently, the phenomenon is due to re-absorption of the fluorescence signals. In their previously reported study of silver by AAS using a conventional hollow cathode lamp Belcher, Dagnall, and West (13) reported an identical upper limit of linearity of 10 ppm in an air-propane flame. However, the working range for the previously described AAS method in aqueous solution was 1-10 ppm whereas in AFS the present study gives a (12) G. F. Kirkbright, T. S. West, and C. Woodward, ANAL. CHEM., 37, 137 (1965). (13) R. Belcher, R. M. Dagnall, and T. S. West, Talunta, 11, 1257 (1964). 338
ANALYTICAL CHEMISTRY
Figure 2. Fluorescence emission in flame under irradiation from silver lamp 25 ppm Ag; lamp currents 20/400 mA; slit 50 f i ; max. gain (20)
range of 0.01-10 ppm-Le., a hundredfold lower limit of determination. The detection limit (1% scale deflection) found for aqueous solutions and the high intensity hollow cathode lamp in the present AFS study was 0.0017 ppm in the air-propane flame at 3280.7 A; the signal to noise ratio was 2 : l . Previously reported data for a 150 W xenon-arc e5citation in an airpropane flame gave 0.35 pprn at 3280.7 A and 0.15 ppm for air-hydrogen (2), while 0.08 was recorded for the xenon arc (150 W) and an oxy-hydrogen flame using a slightly different method of computation and a turbulent (not premixed) flame ( I ) . Following extraction of silver (13) as its di(n-butylamine) salicylate into isobutyl methyl ketone by the method of Betteridge and West (14,we were able to establish calibration curves in the range down to 0.0005 ppm in aqueous solution with a detection limit of 0.00004 ppm as a result of the operation of favorable phase ratios and the more efficient nebulization of the organic solvent. Effect of Foreign Ions. An extensive examination of the interfering effects of about 40 ions on the atomic absorption method for silver was reported in a previous communication (13). Because AFS depends on the same basic phenomena as AAS, it was decided only to examine the potential interference of a selected number of ions in the present study, but to examine these at a 1000-fold excess concentration-Le., 1000 ppm of the foreign ion in the presence of 1 ppm of silver. No interference was observed from Ca, Cu, Fe, Hg, K, Na, Pb, and Zn, indicating also a freedom from scatter of the incident radiation. A slight depression of the silver signal was observed in the presence of 500 ppm of aluminum, but near(14) D. Betteridge and T. (1962).
s. West,
A n d . Chirn. Acta, 26,
101
linear calibration curves could still be obtained for varying amounts of silver in the presence of an approximately constant 500 ppm of aluminum, thus indicating a matrix effect probably associated with decreased atomization of silver from AlzOaparticles in the flame. Atomic Absorption of Silver. Because the apparatus may be used similarly for AAS studies, the source was aligned along the optical axis of the monochromator and the emission burner-head was replaced by a long slot burner. The apparatus was then used to compare air-propane, air-acetylene and nitrous oxide-acetylene for the atomic absorption of silver and to study the effects of lamp currents, etc. The results shown in Table I11 indicate that the best sensitivity is obtained with maximum secondary and low primary cathode currents in an air-propane flame with scale-expansion ( X 5). Lower sensitivity was obtained in a nitrous oxide-acetylene than in an air-acetylene flame even allowing for the shorter length ( 5 cm) of the former flame. Good calibration curves were obtained in the range 0.1-10 ppm. CONCLUSIONS
Measurement of the atomic fluorescence signal of silver in an air-propane flame under irradiation from a high intensity hollow cathode lamp is shown to provide an extremely sensitive and relatively interference-free method for the determination of subnanogram amounts of silver. The range of linearity of calibration curves extends over a 103-fold concentration range from 10 ng/ml upwards to 10 pg/ml in aqueous solution or from ca. 5 X 10-l ng/ml upwards following extraction into isobutyl methyl ketone. No interference was found in the determination of silver by atomic fluorescence in the presence of 1000-ppm concentrations of several ions including iron. Elements such as aluminium which form refractory oxides lower the sensitivity of the AFS pro-
cedure, but do not prevent the determination and can be tolerated if due allowance is made for the matrix effect in drawing up calibration curves. No problems arising from scattering of the incident light were observed in this study. It may also be concluded that for the purposes of analytical atomic fluorescence where a chemically specific source such as the high intensity lamp is used, or a microwave-excited electrodeless discharge tube, the most sensitive results of all might be obtained by simple use of a narrow bandpass or interference filter instead of a monochromator which is subject to considerable signal attenuation. In a few cases there is a possibility that other elements might then be excited in the flame due to spectral cross-matching as has been demonstrated recently in this laboratory with iodine and bismuth at 2062 (15). This is likely to be rather a rare occurrence, however. Under the same instrumental and experimental conditions (difference only in burner head), the atomic-fluorescence procedure described here is about 80 times more sensitive than the absorption method. It is also interesting to note that the best results are obtained in the relatively cool air-propane flame and that the nitrous oxide-acetylene flame used in the fuel-rich mode commonly recommended for atomic absorption measurements of refractory metals such as molybdenum, aluminium, etc., or in the hotter fuel-lean mode is not at all suitable for atomic absorption measurements of silver.
A
RECEIVED for review July 10, 1967. Accepted October 16, 1967. We are indebted to the Science Research Council for financial aid in support of this investigation. (15) R. M. Dagnall, K. C. Thompson, and T. S . West, Tuluntu,
in press.
Microdetermination of Calcium in Biological Material by Automatic Fluorometric Titration Andre B. Borle and F. Norman Briggs Department of Physio/ogy, University of Pittsburgh School of Medicine, Pittsburgh, Pa. 15213
A method is described for the determination of very small amounts of calcium in biological material by automatic fluorometric titration of a calcium-calcein complex with EDTA. The method does not require the separation of calcium. It is very rapid, the titration of 25 nmoles Ca (1 pg) requiring only 2 minutes. The titration is done in a Turner fluorometer and recorded on a milliampere recorder. Delivery of microliters of the EDTA titrant is made by a modified lambda pump and mixed by a specially designed microstirrer placed in the fluorometer. The method is extremely sensitive and can detect 1 nmole (0.04 pg) Ca with a coefficient of variance of less than 2%. It is highly specific and free of interference since ma nesium, phosphate, and ATP do not interfere up to mo ar ratios of 200, 20,000, and 40,000, respectively. Proteins such as albumin, troponin, tropomyosin, actin, and cell sonicates do not interfere with the titration up to weight ratios of 36,000.
B
MOST METHODS available for the determination of small amounts of calcium in biological material suffer from interference by many substances present in the sample. Sodium, potassium, and phosphate interfere with flame photometry
( I , 2); sodium, potassium, phosphate, and proteins with atomic absorption (3); and magnesium, ATP, and nonspecific fluorescent substances with direct fluorometry (4, 5). Complexometric titration of a fluorescent calcium-calcein complex seems to be one of the best methods since the absolute level of fluorescence is unimportant in the determination of the equivalence point (4). Furthermore, if the titration can be shown to be unaffected by the most common interfering ions, the separation of calcium by precipitation and/or elimination of organic material by ashing would become unnecessary. These are steps in which calcium loss or calcium contamination by reagents can occur, and their elimination would be an important improvement. (1) P. S. Chen, Jr., and T. Y. Toribara, ANAL. CHEM., 25, 1642 (1953). (2) R.P. Geyer and E. J. Bowie, Anal. Biochem., 2,360(1961). (3) J. B. Willis, Spectrochim.Acta, 16,259 (1960). (4) D. F. M. Wallach and T. L. Steck, ANAL. CHEM.,35, 1035 (1963). (5) H.B. Collier and G . Duchon, Anal. Biochem.,15,367 (1966). VOL. 40, NO. 2, FEBRUARY 1968
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