ANALYTICAL EDITION
INDUSTRIAL AND ENGINEERlNG HARRISON E. HOWE,EDITOR
Quantitative Spectrographic Determination of Lead in Biological Material JACOB CHOLAK, Kettering Laboratory of Applied Physiology, University of Cincinnati, Cincinnati, Ohio
A
N E A R L I E R p a p e r (1)
ence with the samples, with reA quantitative spectrographic method spect to their salt composition. from this laboratory defor the determination of lead in various The v a r i o u s types of samples scribed a spectrographic method types of biological materials is described, met with in general work may for the quantitative determinain which the quantities of lead introduced be separated for practical purtion of llead i n u r i n e . This into an arc are measured microphotoposes into three g r o u p s , t h e m e t h o d d e p e n d e d upon the analysis of which necessitates measurement of the lengths of metrically in terms of relative lead-line the use of separate lead standthe wedge-shaped lines produced intensities. Some typical results obtained a r d s a n d separatle calibration on a photographic plate by the with known amounts of lead demonstrate curves. i n t e r p o s i t i o n of a rotating an accuracy of *0.00002 mg. for amounts of Each working curve (Figure logarithmic sector (9) between 0.00002 to 0.0004 mg. of lead within the arc. 1) was d r a w n f r o m t h e data the arc and the spectrograph obtained from ten plates, each slit The use of a microphoThe influence of certain variations in of w h i c h s h o w e d five spectra tometer to measure line intensicomposition of samples is illustrated, and of 0.2-cc. portions of each of ties has increased the accuracy other principal sources of error are menthree l e a d s t a n d a r d s . The and flexibility of the method, tioned. mean values for the intensity thereby justifying a description ratios-Pb l i n e X2833.2/ of the" t k c h n i c a s it has been standard of intensity (BiX2898.1) 2, 3, ?'-for each standard applied to the analysis of a wide variety of biological materials. were plotted against the corresponding lead concentrations and the corresponding amounts of lead introduced into the Apparatus arc. The three standards for curve I contained, respectively, The spectrograph and accessory apparatus have been deper 100 cc. of solution, 0.01, 0.10, and 0.20 mg. of lead, 1 mg. scribed prleviously (1). A Bausch and Lomb microphotomeof bismuth (the "internal standard", 7), and the quantity of ter of recent design was employed to measure the line intensalts present in the ash of 1liter of average normal urine. sities. A concentrated stock solution employed for making the standards for curve I and also for use as a diluent for certain samples, Calibration Curves as indicated later, was made by dissolving the following salts: Unknown quantities of dissolved lead which are to be de170.5grams of sodium chloride, 63.5grams of potaseium chloride, 31.5 grams of calcium chloride (CaCl2.6HeO), 20.0 grams of termined spectrographically must obviously be compared magnesium chloride (MgC12.6Hz0), and 37.5 grams of sodium with known quantities of lead in solutions of like composition dihydrogen phosphate (NaH2P04.HgO), in 1 liter of distilled (within certain limits) with respect to the variety and conwater. After gassing with hydrogen sulfide and filtering to remove lead, the filtrate was acidified with triple-diistilled hydrocentration of the associated salts. The lead may be removed chloric acid (silica still), boiled to expel hydrogen sulfide, and from the solution of any ashed biological sample as the sulfide, made u again to 1 liter with triple-distilled water. Each 50 and added to a standard salt solution which is used to make cc. of t&s solution contained sodium, potassium, calcium, magup the known lead standards. Such a procedure has the nesium, and phosphorus in amounts Corresponding to those advantage of its general applicability, but is to be avoided in found in the ash of 1 liter of normal urine (6). (Sulfur was purposely omitted, since its presence or absence was found to be dealing with the minute quantities of lead which occur in without effect upon the spectral intensity, within the limits of biological material, since the manipulations entail unavoidthe concentrations employed.) able loss of' lead and increase the likelihood of contamination. Moderate variations in the composition and concentrations The standards for curve I1 differed from those for curve I of the associated salts do not cause significant variations in only in that each contained 50 mg. of iron per 100 cc. (the results (Table I), and it is best, when feasible, to minimize amount present in 100 cc. of blood). This modification the manipulation of samples by adapting the standard lead followed from the observed influence of iron upon the leadsolutions so as to bring them into approximate correspondline intensity (Table I). 287
288
INDUSTRIAL AND ENGl NEERING CHEMISTRY
The stock solution used in association with curve I1 was prepared just like that for curve I except for the final addition of 1.00 gram of iron in the form of pure iron wire. The standards for curve I11 contained the metals present in the ash of 1 kg. of mixed food, in addition to lead and bismuth in the previously indicated quantities. This type of standard, and the resultant curve, were found t o be necessary in dealing with large composite food samples with their relatively high phosphorus and potassium content.
VOL. 7 , NO. 5
triple-distilled water; the solution of bone was made to contain the ash of 1 to 5 grams of bone per 100 cc., that of blood the ash of 1 gram of blood per 1 cc., and that of other samples excepting feces, the ash of the entire sample (up to 100-gram sample) per 100 cc.; in the case of feces, the adjusted solution contained 1 gram of ash per 100 cc.
The stock solution for making the standards and for use as a diluent was made by dissolving 58.9 grams of calcium chloride, 113.6 grams of magnesium chloride, 37.2 grams of sodium chloride, 174.4 grams of potassium monohydrogen phosphate (KzHP04), and 12.2 grams of potassium chloride, in 1 liter of distilled water. After removing the lead by gassing with hydrogen sulfide and filtering, the filtrate, Licidified with hydrochloric acid, mas boiled to expel hydrogen sulfide, 0.48 gram of iron as pure wire was added, and the volume was brought up to 1 liter with tri le distilled water. Each 50 cc. then contained the amounts o f sodium, potassium, calcium, magnesium, phosphorus, and iron which had been calculated from Lusk's data (4) as present in 1 kg of average mixed food materials. TABLE I. EFFECTOF VARIATIONS
Vaiiation from Standard' One-half average concenhrahon
T&&
average concentra-
tion None 100% increase in NaCl Potassium absent 100% increase id potasaium Phos horic acid absent 300f increase in phosp oric acid Calcium absent, 100% jncrease In calcium iMagneaium absent 100% increase in magnesium Sulfuric acid absent Addition of sulfuric acid (7 5 grams) 100% increase in NaCl and 50% decrease in potaa-
Lead Lead Lead from from from StandStandStandRatio ard Ratio ard Ratio ard Found Curve Found Curve Found Curve
Mo.
Mo.
Mo.
0.51
0 01
166
0 10
2.76
0.49 0.39 0.48 0.40 0.51 0.65
0 0 0 0 0 0
01 01 01 01 01 02
1.46 1.68 1.69 1.61 1.70 1.60
0 09 0.10 0 11 0 10 0 11 0.10
2.90 0.20 2.93 0.20 3.05 0.21 2.69 0.18 2.63 0.18 2.63 '0.18
0.53 0.39 0.53 0.35
0 0 0 0
01 01 02 01
1.56 1.45 1.65 1.58
0.10 0.09 0.10 0.10
2.16 2.75 2.74 2.83
0 55 0 39
0 02 0 01
1.81 1.68
0.11 2.66 0.10 2.93
0.18 0.20
0.55
0 02
1 71
0 11
2.55
0 17
2.80
0 19
2.73
0 18
2.70
0 18
2 56 2.42
0 17 0.160
slum 0.33 0 01 1.64 0.10 100% increase in potassium and 50% decrease in - 0.62 0 02 1.61 0 10 NaCl 100% increase in NaCl and 50% decrease in phos0.53 0 02 1.56 0 10 phorio acid 1007 inorease in pbosp h k c acid and 50% decrease in NaCl 0.54 0 02 1.52 0 09 Standard plus 60 mg. Fe 0.50 0.01 1.60 0 10 d Mean values for calibratioh curve I: 0.01 = 0.47 i0.01, standard deviation A 0.10 0.10 = 1.61 i0.015, standard dev!at/on A 0.15 0.20 = 2.95 i0.024,standard deviation i0.25 0 From 30 spectra. 1, From 15 spectra.
0 20
0.145 0.19 0.19 0.19
Curve I was used in the analysis of urine, bone, fat, nervous tissue, spinal fluid, and individual food samples or others low in iron; curve I1 for the analysis of blood, liver, spleen, heart, muscle, and individual foods or other samples which contained appreciable amounts of iron; and curve I11 for the analysis of large composite samples of food.
Preparation of Samples for Arcking URINE. Samples were prepared as previously described ( I ) , except that 0.1 mg. of bismuth per 10 cc. of final volume was used as the internal standard. OTHERBIOLOGICAL SAMPLES.Individual items of food (but not mixed food samples), blood, and animal tissues with the exception of bone, were digested with sulfuric acid and triple' distilled nitric acid in uartz Kjeldahl flasks, and evaporated to dryness. Because their high calcium content bone and feces were ashed in a muffle furnace at 500' C. the former having undergone an initial digestion with triple-dstilled nitric acid. The ash of each sample was dissolved in triple-distilled hydrochloric acid and the final volume was adjusted by dilution with
07
FIGURE 1. VARIATION OF INTENSITY RATIOS PbX2833*2WITH LEADC,OSCENTRATION BiX2898.1 These materials, to which none of the calibration curves applied directly, were adapted to analysis with either curve I or curve I1 by the admixture of their dissolved ash and the "leadfree" stock solution used in the preparation of the standards for curves I and 11, as follows: Two cubic centimeters of the solution of the sample and 2 cc. of the appropriate stock salt solution (to which had been added 1 mg. of bismuth per 100 cc.) were introduced into 15-cc. Pyrex centrifuge tubes and concentrated to 2 cc. Each 0.2 cc. arcked then contained approximately 35 mg. of the inorganic salts of the stock solution plus a minute and practically unimportant quantity of ash derived from the sample. ("Lead-free" stock solution is one that has been treated previously with hydrogen sulfide and contains not more than 0.002 mg. per 100 cc. The quantity of lead in'a few cubic centimeters of such a solution may be ignored, since the lowest lead standard contains 0.01 mg. per 100 cc.) SPINALFLUID.To 10 cc. of spinal fluid in a small quartz dish were added 2 cc. of triple-distilled nitric acid. After evaporating to dryness, the residue was ashed in a muffle furnace. The ash was taken up in a few drops of triple-distilled nitric acid and a little triple-distilled water, rinsed into a 15-CC.Pyrex centrifuge tube, and evaporated to 2 cc. in the water bath after the addition of 2 cc. of the stock solution used for the standards of curve I (to which had been added 1 mg. of bismuth per 100 cc.). Each 0.2 cc. then represented 1 cc. of spinal fluid. MIXED FOODSAMPLES.Composite food samples were digested with nitric acid and ignited in a muffle furnace at a temperature of 500' C. (High-grade nitric acid without redistillation and Pyrex glass vessels may be used here. The contamination from their use is insignificant, in view of the considerable amounts of lead usually present in food.) The residue was dissolved in the least sufficient amount of triple-distilled nitric acid and triple-distilled water, transferred to a graduated glassstoppered 100-cc. Pyrex cylinder, and filtered if necessary to remove carbon. The proper amount of bismuth was then added and the volume of the solution adjusted so that 1 to 1.5 grams of ash were present in each 10 cc. of solution. Each 0.2 cc. of the solution then contained 20 to 30 mg. of inorganic salts.
Spectrographic and Photometric Technic Employing successively the same 0.1-cc. capillary pipet, 0.2cc. portions of the prepared solutions were placed in the craters of electrodes (7 mm. Acheson regraphitized graphite, 40 mm. long with a crater 3 mm. wide and 10 mm. deep). After drying for several hours at 48.89' C. (120" F.) the electrode with the crater was made the negative, and for each such electrode a fresh electrode was made the positive in the arc. The electrodes,
SEPTEMBER 15, 1935
ANALYTICAL EDITION
properly centered, were separated 5 mm. and the arc was started by momeintarily bridging the gap with a graphite rod. Threeminute exposures were provided with an arc of 10 amperes and a 60-volt drop across the terminals. After passing through a quartz condensing lens the radiations, reduced 50 per cent by a rotating sector, fell on a slit 5 mm. long and 0.04 mm. wide. This procedure yielded a light background on the photographic plate, as well as lines of such width and length, when projected, as to cover completely the slit of the microphotometer. Five spectra of each of three samples were photographed on each plate (10 X 25 cm., 4 X 10 inches, Eastman No. 33). The plates were developed for 7 to 8 minutes at a temperature of 16.67' C. (62' F.) in a mechanically agitated bath of alkalinehydroquinone developer, which was changed for each plate. After fixing (15 minutes) and washing (30 to 45 minutes) the developed plates were wiped gently with clean chamois and dried at room temperature for several hours. Microphotometer measurements were carried out on thoroughly dried plates only. The throw of the galvanometer for the emulsion adjacent to each line was taken as the zero reading and the differences between these readings and those for the lines themselves were recorded as the line intensities. The ratio, reading for Pb line X2833.2/reading for Bi line X2898.1, was then recorded for each spectrogram, T h e value for the average ratio of the five spectra of each sample was then interpolated from the appropriate standard curve (Figure 1).
289
must be cleaned with hot nitric acid, followed by hot tripledistilled water, before each period of use. Samples once started should be carried to completion as rapidly as possible to avoid possible contamination. Various other opportunities for contamination have been discussed (8). TABLE11. RESULTS WITH KSOWNQUANTITIES OF LEAD Pb Added
Curve Used
Ratio Pb/Bi
Mean
Value
0.05
Me.
MU.
I
0.01 0.01 0.02 0.02 0.01
0.01
I1
1.04 1.06 1 .oo 0.98 1.05 1.13 1.05 Av. 1.07
0.06 0.06 0.05 0.06 0.06 0.06
0.06
1.61
1.52 1.42 1.75 1.56 1.70 Av. 1.59
0.09 0.08 0.11 0.10 0.11
0.18
0.10
0.12
I
1.75
1.64 1.79 1.84 Av. 1.75
0.10 0.11 0.12
0.I1
0.12
I
1.76
1.59 1.79 1.70 Av. 1.70
0.10 0.11 0.11
0.11
0.20
I
2.95
2.86 2.86 2.89 2.79 2.68 Av. 2.81
0.20 0.20 0.20 0.18 0.18
0.19
Discussion I n Table I1 are recorded the results obtained with known quantitie;v of lead added to standard stock salt solutions. Although the repeated observations yielded identical results in several instances, successive spectra of the same sample may vary as much as 20 per cent. The lack of closer agreement is due primarily to the method of excitation employed. The arc between graphite electrodes has a tendency to wander and it is almost impossible therefore to obtain identical excitation conditions for successive spectrograms. The stability of the arc may be increased to some extent by reversing the polarities of the electrodes (b),as compared with the usual procedure, and by arcking relatively large quantities of maIf the arc begins to wander, however, terial, 15 to 50 mg. it becomes very difficult t o center it on the slit, and if most of the wandering occurs while either the lead or the bismuth is being volatilized into the arc the spectrogram will not correspond to those of the same sample which were made when the arc was consistently centered. The increased accuracy which results from the use of the average value from a number of spectra of the sample is illustrated in Table 11. In general the averages of five observations give results which rarely vary as much as 0.01 mg. from the amounts known to be present. The data of Table I show that variations in the concentrations of iron and phosphates result in significant variation in the values obtained for lead concentrations above 0.10 mg. of lead per 100 cc. This variation is remedied, in the case of iron, b y applying the special curve derived for the analysis of iron-containing samples. When appreciable quantities of phosphoric acid occur in samples (easily determined from the intensities of the phosphorus lines on the plate), the sample should be diluted with stock salt solution in order to bring the amount of lead below 0.10 mg. per 100 cc. and thus to permit the application of the lower and more stable part of the curve. Occasional samples contain bismuth in such amounts as to exclude its use as the internal standard. Such samples may be analyzed without the use of the internal standard by a procedure similar to that previously described (1). The principal sources of error, other than the factors cited above, arise from contamination of the sample during the process of' collection and preparation. All vessels employed
Pb Recorded
0.42 0.44 0.47 0.52 0.54 0.42 Av. 0.47
Mg.
0.01
Pb Found
In dealing with biological materials the choice of lead line is limited to the most persistent line (X2833.2) since it is frequently the only line to appear. With amounts of lead ranging from 0.00002 to 0.0004 mg. (0.01 to 0.20 mg. per 100 cc.) in the arc, the intensities of the lead lines produced in the photographic plate are such as to fall on the initial part of the blackening curve for the line X2833.2. In this region the proportionality between the intensity (blackening) of the line and the intensity of radiation (concentration of lead) is more nearly expressed by the approximate formula blackening
=
constant X i
than by blackening
=
gamma log i/io
which holds for the straight-line portion of the blackening curve (3, page 14). It is more practical and convenient, therefore, to plot the intensity ratios against concentration instead of log concentration, in the development of standard calibration curves for the range of concentration dealt with herein. Lead concentrations above the range to which the curves apply may be adapted by dilution with the appropriate solution of salts; otherwise it is necessary to apply the observed data to calibration curves which are based upon relationships obtaining a t a higher range of lead concentrations.
Summary The technic as described has the following advantages over a method described in an earlier paper (1): It has an accuracy of kO.01 mg. over the entire range of applicability-i. e., for amounts of lead between 0.01 and 0.20 mg. It permits the use of a single set of standard calibration
INDUSTRIAL AND ENGINEERING CHEMISTRY
290
curves for interpolation of results, thus saving the time and plates required for making individual curves from the results on each plate.
Literature Cited (1) Cholak, J., J . Am. Chem. Soc.. 57, 104 (1935). (2) Gerlach, w., and Schweitser, E., “Foundations and Methods of Chemical Analysis by the Emission Spectrum,” London, Adam Hilger, Ltd., 1929. (3) Lundegardh, H.,“Die quantitative Spektralanalyse der Elemente,” P a r t 11, Jena, Gustav Fischer, 1934.
VOL. 7, NO. 5
(4) Lusk, G.,
“Science of Nutrition,” 3rd ed., p. 360, Philadelphia, W. B. Saunders Co., 1919. (5) Mannkopff, R., and Peters, C., 2. Phgasik, 70,444 (1931). (6) Mathews, A. P., “Physiological Chemistry,” 5 t h ed., pp. 730, 1186, 1187, New York, Wm. Wood and Co., 1930. (7) Nitchie, C.C., and Standen, G. W., IND.ENG.CHEM.,Anal. Ed., 4, 182 (1932). (8) RabinowitCh, 1. Dingwall, A.8 and Maokay, F. H.9 J. Bid. Chem., 103, 707 (1933). (9) Scheibe, G., and Neuhausser, A., Z.angew. Chem., 41,1218(1928).
RECEIVED March 18, 1935.
Volumetric Determination of Evaporation Rates L. A. WETLAUFER AND J. B. GREGOR E. I. du Pont de Nemours & Co., Inc., 3500 Grays Ferry Ave., Philadelphia, Pa,
H E development of modern protective coatings, parnounced by Lowell (4, called the Evap-0-Rotor, requires titularly since the introduction of many of the now the usual analytical weighings. It employs a turntable with common synthetic resins, has emphasized the imporcapacity for several samples, the table being protected tanceof the evaporation rates of the solvents and thinners by a shield several inches high to minimize the effect of used in their manufacture and application. A greater variety stray air currents. The table slowly rotates, establishing a of materials, a more rigid control of finished product properconstant air flow which removes the heavy vapors from the ties, and the development of fast-drying enamels have all evaporating surfaces. This method is believed to be one of the most satisfactory of the gravimetric procedures developed been contributing factors. All of the present methods for determining evaporation to date in that a number of samples may be evaporated sirates with which the authors are familiar are gravimetric. In multaneously, and that a simple method of air regulation is the usual procedure 1 to 2 grams of the liquid are placed in a employed. For the slower evaporating liquids, however, the tared dish such as a friction-top can cover and weighed a t intertime and labor factors are undesirable for routine control vals during the period of evaporation. Those which employ work. a special balance with sample suspended, thus doing away At best any method for measuring the rates of evaporation with tedious analytical weighings, are open to the criticism of liquids is not a precise criterion of how these liquids will that only one or two samples can be measured at a time. affect the drying time of finishes. Many factors which affect Perhaps the best known of these special instruments is the evaporation rates will affect drying time in different ways. Durrans (1)points out that vapor pressure, heat conductivity, Hart evaporation balance (g), a device equipped for one samlatent heat of evaporation, molecular association, surface ple. Another special instrument, the Jolly balance (S), is equipped for two samples but, according to the method, retension, and humidity are important considerations. He quires a determination of a standard at the same time as the points out further that in mixtures of liquids the molecular attraction of one component by another and the depression sample. It would be necessary, therefore, to use several units of these special instruments in order to conduct a numof the vapor pressure of one component by another are likeber of tests at one time, thus involving a considerable investwise governing factors. Solvent power and vapor density should be added to the list. A study of the comparative ment in equipment. Another difficulty encountered with most gravimetric rates of evaporation is therefore of value only in a general methods is the control of air flow over the exposed area of the sense. In the formulation of a product, selection of likely solvents or thinners can, of course, be made from graphs sample. Wilson and IVorster (6) attempted to overcome this feature by the use of a special tunnel in which the samples which express these data, but the materials so chosen must be thoroughly evaluated in the are placed and through which formula in order to estaba constant flow of air is conlish the one or the blend ducted, This procedure, of course, r e q u i r e s m a n u a l which most nearly satisfies the p r o p e r t i e s r e q u i r e d . analytical weighings a t interThe most specific value to vals, and is one of the things be gained from evaporation which the special balances, data appears to be that of such as the Hart and the c o n t r o l l i n g the uniformity Jolly, overcame by making of a given liquid as received the weighings a u t o m a t i c . Also in t h e Wilson a n d by the c o n s u m e r , a n d of Worster method it is adb l e n d s which may be premittedly difficult to control p a r e d prior t o manufacture. the current of air with a good With these things in mind degree of a c c u r a c y . AnFIGURE 1. DISTILLATION CURVES t h e a t t e m p t to establish a o t h e r device recently an-
T
,
