Sorthwestern Cniversity, Kov. 16, 1945. (8) DeVaney, F. D., Ind. Eag. Chem. 39, 26 (1947). (9) DuBois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., Smith, F., ANAL.CHEM.28, 350 (1956). (10) Flaschka. H.. Chemist Analvst 44. 2-7 (1955).‘ (11) Flaschka, H., Abdine, H., Zbid., 44, 30-1 (1955). (12) Grzhivo, V. S., Sazich. Chteniya 1962 1953 , 78-90. (13) Kerr, R. W.,“Chemistry and Industry of Starch,” pp. 659-72, Academic Press, Pr’ew k’ork, 1950. (14) Love, D. L., Ph.D. thesis, Pennsylvania State University, University Park, Pa., 1955. (15) Mackinney, G., Temmer, O., J . Am. Chem. SOC.70, 3586 (1948). (16) Meites, L., Meites, T., SAL. CHEM. 20, 984 (1948). (17) Middendrop, J. A4,, Rec. trac. chim. 38, l(1919). (18) Mitchell, D. R., ”Coal Preparation,” pp. 609-47, Am. Inst. Mining,
LITERATURE CITED
(1) Bates, F. J., “Polarimetry, Sacchar-
(2)
(3)
(4)
(5)
(6) (7)
imetry and Sugars,” Government Printing Office, Washington, D. C., 1942. Brautlecht, C. .4., “Starch-Its Sources, Production and Cses,” pp. 330-53, Reinhold, Sew York, 1953. Browne, C. A., Zerban, F. R., “Physical and Chemical Methods of Sugar Analysis,” 3rd ed., pp. 1124-33, Wiley, New York, 1941. Cantor, S. M., Peniston, Q . P., J . A m . Chem. SOC.62, 2113 (1940). Chang, C. S., Cooke, S. R B., Huch, R. O., Trans. Am. Inst. JIznzng M e t . Engrs. 196, Mznzng Eng. 5, KO.12, 1282 (1953). Clendenning, K. il , Can. J . Research C20, 403 (1942); B23, 113, 239 (1945); F26, 185 (1948). Dean, G. R., Peniston, Q . P., Cantor, S. M., Technical Conference,
.
I
Met., Petrol. Engrs., S e w l-ork, 1954. __._
(19) RIorris, D. L., Science 107, 254 ( 1948). (20) Radley! J. S., “Starch and Its Derivatives,” Vol. 2, 3rd e d , pp. 35686, Chapman & Hall, London, 1953. (21) Schulz, S . F., Cooke, S. R. B., Ind. Eng. Chem. 45, 2767 (1953). (22) Sun, 8.C., “Effect of Organic Flocculants on Coal Sedimentation,” Chicago Meeting, -4m. Inst. Nining, Met., Petrol. Engre., February 1955. (23) Turner, J. H., Rebers, P. A , , Barrick, P. L., Cotton, R. H., A s . 4 ~ CHEX. . 26, 898 (1954). (24) Van Dyk, J. W., Caldwell, 11. L., Ibid.,28, 318 (1956). (25) Viles, F. J., Jr., Silverman, L., Ibid., 21,950 (1949). RECEIVEDfor review August 16, 1957. Accepted February 3, 1958. College of Mineral Industries Contribution So. 57-54.
Condensed Direct Current Arc Excitation for Spectrochemical Analysis of Plant Materials I
H. E. BRAUN Department of Chemistry, Ontario Agricultural College, Guelph, Ontario, Canada
b A method is described for the simultaneous quantitative determination of calcium, magnesium, phosphorus, iron, manganese, copper, and boron in plant tissue. An acidic aqueous solution of the plant ash is impregnated into powdered graphite held in a cratered electrode. The electrode is dried and then excited by means of a condensed direct current arc. A comparison of spectrographic and chemical analyses of various samples within several plant species indicates that the accuracy of the method is adequate for routine quantitative analysis of plant tissue and the reproducibility of the method is excellent.
S
analyses of plant materials for the major elements calcium, magnesium, and phosphorus are generally obtained by direct excitation of plant ash or plant ash solutions, as chemical concentration is seldom required. Because i t is adaptable t o routine analysis, the copper electrode procedure of Fred, Nachtrieb, and Tomkins (3) was investigated in this laboratory. Sample solution residues were mounted on flat opposed copper electro es and excited by means of a higR voltage alternating current spark. These investigations revealed that large discrepancies between chemical and spectrographic analyses for calcium, magnesium, and phosphorus PECTROGRAPHIC
1076
ANALYTICAL CHEMISTRY
were prevalent, presumably as the result of the high salt concentrations encountered in most plant materials as noted by Feldman ( 2 ) . A related disadvantage of the copper spark technique is the need for preparing characteristic sets of standard concentration curves for each particular species of plant material under investigation because of the dependence of emission characteristics on the concentrational levels of the constituent major ash elements. Consequently, one established set of working curves cannot be applied to the analysis of a variety of plant species, as in agricultural research. I n the search for a method which would equal the convenience of the copper electrode procedure, investigations with a direct current condensed arc type of excitation revealed the possibilities of overcoming the above disadvantages. This paper describes the technique developed to permit simultaneous quantitative spectrochemical determinations of calcium, magnesium, phosphorus, iron, boron, manganese, and copper from one established set of standard concentration curves by means of condensed direct current arc excitation of plant ash solutions. CHEMICAL PROCEDURE
Purification of Reagents.
trated
C.P.
Concenhydrochloric acid ( 1 2 N ) is
diluted n i t h a n equal part of n-ater and t h e mixture is distilled through a n all-glass distillation apparatus. The strength of the purified acid is very close to 6 S . This is the only reagent required by the method which needs to bepurified: Internal Standard Solution. Cobalt serves satisfactorily as t h e internal standard. Under t h e conditions of this method, the trace amounts of cobalt present in plant material are not detectable. A standard cobalt solution IS prepared by dissolving 4.939 grams of reagent grade cobaltous nitrate [Co(N03)2.6HzO]in 100.0ml. of water to yield a concentration of 10.0 mg. of cobalt per ml. To 80 nil. of redistilled 6 S hydrochloric acid, 4.0 ml. of the standard cobalt solution are added, and the total volume is adjusted to 100.0 ml. with water. Preparation of Standards. Individual standard stock solutions of t h e elements to be determined are prepared from analytical reagent salts and pure metals. B y using appropriate amounts of these solutions, a composite stock solution is prepared so that 2.0 nil. of final solution contain a concentration of: Mg. Potassium Calcium Magnesium Phosphorus
25
15 3 3
Y
Iron
Boron Manganese Copper
150 50
25 20
This composite solution contains the
3
I
polarity switch
I
i
r.4
1 I
.Ol
1
,
8
t
I
8
8
l
IO
, ,
I 8 l i i U
0
MILLIGRAM
.-
1b-l
CONCEhT?ATION
Figure 2. Analysis curves for copper, manganese, iron, boron, magnesium, phosphorus, and calcium based o n Co 3044 as internal standard Figure 1. Circuit diagram of source unit with condensed direct current arc modification listed elements in approximately the same proportions as present in a solution of plant ash. Four series of standards of various concentration levels are then prepared by making the following additions to 2.0 nil. of the composite stock solution:
Calcium standards 3[agnrsiuni standards Phosphorus standards
Ng. 1.5, 30, 45 3 . 6, 9 3 , 6, 9 -i
;\[inor elemexit standards Iron Boron hlanganese Copper
150, 300, 450 50, 100, 150 2 5 , 50, 75 20, 40, 60
S e x t , 2.0 nil. of 6 S hydrochloric acid and 2.0 mg. of cobalt as internal standard are added, and the final yolume is adjusted to 5.0 ml. Separate series of standard solutions for the three major elements are prepared to minimize possible effects of high salt concentrations such as would occur if the additions of calcium, magnesium, and phosphorus were included in one composite standard solution. Treatment of Plant Material. Airdried plant tissue is finely ground and then ashed a t 450' C until a11 organic matter has been removed. Then 0.200 gram of the plant ash is dissolved in 5.0 ml. of the internal standard solution (equivalent to 2.0 nip. of cobalt and 4.0 nil. of 6 S hjdrochloric acid) and filtered through Khatmun KO. 5 filter paper. I n the estimation of the size of ash sample to be used. 200 nig. of plant ash produced satisfactory spectrum densities for the element concentrations normally encountered in plnnt tissue. Smaller amounts of ash rewlted in d f i c u l t y in obtaining accurate transmittance readings for weak phosphorus and boron lines, while larger than 0.2gram samples tended to produce correspondingly hearier magnebiuni lines.
SPECTROGRAPHIC PROCEDURE
Apparatus. Spectrograph, Applied Research Laboratories' 1.5-meter concave grating spectrograph. Excitation source, Technical Sewice Laboratories' (Toronto, Ontario) spectro.source unit. Microphotometer, Applied Research Laboratories' comparator-densitometer. Recording film, Kodak spectrum analysis S o . 1 35-mni. film. Preparation of Electrodes. Standard grade 0.25-inch graphite rod is cut into 1-inch lengths after being cratered t o a depth of 5 m m . and a diameter of 5 mni. with t h e aid of an A.R.L. electrode cutter. The crater is filled n i t h 45 t o 50 mg. of -200 mesh spectrographically pure nonbriquetting type graphite powder. The method of mounting the sample on t h e electrodes is similar t o the procedure outlined by Mathis ( 5 ) . Approximately 0.1 ml. of sample solution or standard solution is slowly transferred to the top of the filled crater with a micropipet and allowed to absorh partially into the powdered graphite. The electrodes are then dried in a n oven a t 100" C. for a minimum of 4 hours. After drying, the graphite-chloride salt mixture is firmly packed into the bottom of the crater with a piece of graphite rod which has been shaped so that it will just fit the crater diameter. Counterelectrodes are hemispherically pointed Lvith a 20" cone in the -4.R.L. electrode shaper. Blank determinations with standard grade graphite rod and with spectrographically-pure graphite rod did not justify use of the high purity graphite. K i t h the conditionq of the excitation method under invedgation, none of the analytical lines in question showed U ~ I as contaminants with the standard grade graphite. According to Mathis ( 5 ) .the rapidity with which the solution is absorbed into the graphite pon-der n ill influence the general intensity of the spectrum. This n-as found to hold true in this technique. Rapid absorption of the solution into the electrode results in
the production of relatively weak spectra. This is overcome by placing an additional drop of solution on the electrode so that some liquid remains unabsorbed just prior to placing the electrodes in the oven for drying. To ensure a minimum of moisture build-up in the sample, the electrodes are left in the oven until they may be excited immediately. After the required drying period, the graphite-chloride salt mixturp tends to be rather loose and fluffy. Firm packing of the mixture after removal from the oven results in the retention of the sample in the crater for a longer period during the excitation interval and also produces greater uniforniity of spectrum densities from one sample to another. Excitation Conditions. With a n input line voltage of 220, t h e spectrosource is adjusted t o deliver a current of 6.0 amperes across graphite electrodes using t h e conventional diiect current arc discharge. The source unit is then switched over t o the condensed direct current arc circuit, the details of which are shown in Figure 1. Preliminary adjustment of the current is made with the normal direct current arc because no significant ampere reading will register with the condensed arc. A capacitance setting of 7.5 pf. is used throughout the whole series of investigations. The sample electrode and counterelectrode are spaced with an analytical gap of 4 mm. The polarity is such that the lower electrode (sample electrode) forms the anode. d slit width of 40 microns is used. K O filters and no shutters are employed. Grating doors are set a t 4:4. The excitation time is 20 seconds n i t h the full 20-second exposure being photographed. Under the influence of the discharge of the condensed arc, all of the sample is completely sprayrd out of the crater by the end of the 20second excitation period. Film Processing. Films are developed for 3 minutes a t io' F. n i t h Kodak D-19 developer. neutralized VOL. 30, NO. 6, JUNE 1958
1077
for 15 seconds in a stop bath, and fixed for 5 minutes in Kodak prepared rapid h e r and hardener. The film is spraywashed in water, sponge-dried, and finally dried in an infrared film dryer for 1.5 minutes. Photometry. Film calibration is performed by using a two-step splitfield filter in conjunction with t h e iron spectrum. As Kodak SA KO. 1 film has a reasonably constant gamma over
Table I.
Species Alfalfa
Red clover
Timothy
Cauliflower
Celery
Apple leaves
Soybeans
the wave-length range of 2400 to 3300 A. and the analytical lines under investigation are all located within this range, a single calibration curve is used in determining intensity ratios. A single cobalt line, Co 3044, is used as the internal standard reference for the following analysis lines: Boron 2498 Phosphorus 2535
2997 3020.5
3274
of Plant Material
(411 values reported as yo in ash) c = average chemical composition of total samples within each species s = standard deviation between chemical and spectrographic methods n = total number of chemical and spectrographic comparisons Magnesium Phosphorus Iron Manganese Calcium Chem. Spec. Chem. Spec. Chem. Spec: Chem. Spec. Chem. Spec. 0,024 0.022 17.5 17.5 2.45 3.13 2.68 2.56 0.127 0.147 0.025 0.027 0.180 0.206 15.1 15.4 3.03 3.16 2.60 2.56 2.60 2.51 0.027 0.023 21.5 22.7 0.128 0.144 3.46 4.05 0.028 0.023 20.5 23.9 0.097 0.122 2.72 2.51 2.82 3.38 ... ... 2.62 2.58 25.0 24.4 3.55 4.50 0.114 0.137 C = E = 0.129 0.026 E = 19,92 C = 3.06 C = 2.64 s = 0.0041 0.0015 s = s = 0.070 s = s = 1.36 0,296 0.036 0.035 18.6 19.6 4 38 4.93 2.40 2.69 0.098 0.112 0.040 0,041 2.24 2.57 4.12 4.50 0.300 0.270 16.2 15.6 2.62 2.63 0,038 0.032 4.25 4.52 0.146 0.144 18.9 18.5 0.035 0.034 0.150 0.134 2.16 2.21 20.5 19.7 5.43 6.34 0 034 0.035 0.261 0.233 2.33 2.40 16.6 17.6 4.92 5.15 E = c = 0.191 0,037 4.62 € = 2.35 € = 18.16 E = 0.0114 0.0022 s = s = s = 0.148 s = 0.277 0.26 s = 4.12 4.20 0,108 0.093 0.029 0.035 7.7 6.6 2.83 2.95 0.028 0.029 4.12 3.60 0.097 0,099 8.0 6.3 2.68 2.07 0.132 0.122 0.030 0.036 3.88 3.72 8.6 6.4 2.60 2.52 0.035 0.037 4.18 4.10 0.105 0.096 2.73 2.31 9.6 6.3 0,110 0.114 0,028 0.032 4.27 3.89 7.2 2.37 2.31 8.6 € = 0.110 C = 0.030 E = C = 4.11 € = 8.50 2.64 s = 0,0051 0.0025 s = s = s = 0.197 0.245 s = 0.86 0,077 0.069 0.031 0.037 24.2 18.4 2.75 3.02 1.65 1.46 0.044 0.053 0.094 0.084 2.23 2.45 22 4 18.4 1.77 2.19 0,053 0.055 2.12 2.33 0.100 0.090 21.3 20.5 1.73 2.06 0.047 0.054 0.091 0.079 2.38 2.56 22.8 22.5 1.87 2.38 0.038 0.051 2.35 2.22 0.088 0.081 2 03 2.51 23.7 19.8 c= F = 0.043 E = 2.37 0.090 € = € = 1.81 22.88 s = 0.0044 0.0019 s = s = 0.129 s = 0.053 s = 2.33 0.125 0.127 0.017 0.021 3.62 3.60 7.2 0.88 0.68 7.0 0,016 0.021 0.050 0.057 4.44 4.50 1.04 0.82 6.8 6.5 0.114 0.109 0.016 0.018 2.14 2.10 0.77 0.74 14.1 17.6 0.026 0.026 0.075 0.049 2.45 2.18 0.72 0.80 12.6 13.1 ... ... 3.20 2.83 0.051 0.046 0.84 0.82 12.4 13.5 = 3.17 € = c= 0.083 0.019 E = 10.52 E = 0.85 s = 0.0089 0.0022 s = s = 0.159 s = 1.38 0.094 s = 0.021 0.019 2.52 2 . 5 8 0.151 0.137 1 4 . 1 12.9 3.01 3.00 0.024 0,023 0.124 0.128 3.25 3.04 17.8 16.6 3.63 3.36 0.030 0.026 0.140 0.138 2.42 2.47 15.7 14.6 3.00 3.13 0.024 0.026 0.117 0.123 1.88 1.93 3.23 3.26 16.6 14.9 0.043 0.046 2.15 2.11 0.156 0.154 3.02 3.53 13.5 14.0 € = 0.024 E = 2.45 € = 0.138 € = 3.18 € = 15.5 0.0010 s = s = 0.0050 s = 0.072 0.206 s = s = 0.43 0.033 0.034 0.188 0.184 4.30 4.41 10.4 10.9 6.5 5.1 0.027 0.030 0.180 0.162 13.2 13.7 3.40 3.80 5.0 6.4 0.028 0,018 11.3 11.8 0.180 0.183 4.20 3.99 8.1 5.5 0.221 0.220 0.029 0.021 10.0 11.4 3.87 3.95 6.4 4.9 0,041 0.034 0.169 0.165 11.3 12.2 8.4 5.1 4.50 3.98 € = 0.032 E = 0.188 E = 11.2 7.2 € = 4.05 d = 0.0037 0.0068 s = s = s = 0.398 0.190 0.87 s =
ANALYTICAL CHEMISTRY
Copper
K i t h the concentration of the internal standard as described previously, the transniittance of Co 3044 in most cases falls between 15 and 25%. Deviations of the internal standard transmittance beyond either limit of this range do not significantly affect the
Comparison of Spectrographic vs. Chemical Analyses for Seven Species
Standard Deviations as Calculated Ca blg n 35 35 2.90 E 14.7 S 1.35 0 250
1078
Calcium Iron
Manganese 2576 llagnesium 2781
on Combined Total of Samples for Seven Species P Fe Mn B 33 28 35 35 0.031 0 046 0.133 4.03 0.0031 0 0037 0.0086 0.281
Boron Chem. Spec. 0.031 0.029 0.034 0.032 0.037 0.032 0.035 0.036 0.035 0.033 C = 0.034 s = 0.0015 0.032 0.033 0.034 0.032 ... ... ... ... ... ... E = 0,033 0.0014 s = 0.021 0.021 0.018 0.023 0.020 0.020 0.021 0.023
...
€ =
s =
...
0,020 0.0023
0: 028 0.024
...
...
01035 0,029 € = 0.032 s = 0.0014 0.014 0.015 0.016 0.015 0.020 0.018 0.024 0.021 0,023 0.029 0.019 E = 0.0021 s = 0.068 0.069 0.090 0.077 0.086 0.087 0.058 0,060 0.070 0.062 E = 0.074 0.0014 s = 0.084 0.092 0.095 0.109 0.080 0.084 0.073 0.082 0.092 0.097 E = 0.083 0.0029 s =
intensity ratios between the unknown element and cobalt. The densitometer is adjusted so that it reads 100% transmittance for clear film. The per cent transmittance of the analysis lines and the reference line are then recorded and converted to intensity ratios by means of an A.R.L. calculating board. No background corrections are made.
Table II.
Reproducibility on Ten Replicates of Red Clover
Element, % in 8sh Ca Mg P A
Fe, p.p.m. Mn, p.p.m. B, p.p.m. Cu, p.p.m.
Mean 17.6 4.49 2.05 212 40.0
Std. Dev. 0.620
0.213 0.072 13.6
1.49 1.90 1.97
26.1
24.1
Coefficient of Variation, yo 3.5 4.7 3.5 6.4 3.7 7.3 8.2
EXPERIMENTAL RESULTS
Standard Concentration Curves. The working curves established from the standard solutions are shown in Figure 2 . Each experimental point on which the curves are based represents the average of five determinations photographed on five individual films. With the use of the curve Ca 2997 as obtained from the standards, an obvious bias between spectrographic and chemical values was produced. I n order to obtain a representative curve, a series of plant samples covering a wide range of calcium contents was analyzed chemically. These calcium values were plotted against intensity ratios of Ca 2997 for the same samples as obtained by the described spectrographic procedure. The working curve for Ca 2997 was then constructed by the method of least squares and this is the curve represented in Figure 2. The main difference between this curve and the synthetic calcium curve is a marked increase in the slope, with the fiducial points of the two curves occurring a t an approximate concentration of 60 mg. of calcium. Because the calcium curve of Figure 2 is based upon analyses of several different plant species, the author considers the curve valid. The approximate sensitivities and niaximum concentration limits of the analytical lines are as follows: Rlg.
Calcium Magnesium Phosphorus Iron
7 5 t o 150 0.75 t o 20 2 . 5 t o 20 0 078 to 2
Manganese Boron Copper
25 to 500 20 to 500 10 to 150
’
Y
These concentration ranges are broad enough to encompass the analysis of the seven different plant species used in this investigation. The analytical system is flexible to the extent that larger or smaller samples may be prepared with concentrations occurring within the above limits. Accuracy. I n order t o determine the accuracy of the method, individual samples of each of seven species of plant material were first analyzed by chemical methods. Calcium contents were determined by flame photometry;
magnesium was chemically determined by the method of Young and Gill (6); and boron was determined by the method of MacDougall and Biggs (4). Phosphorus, manganese, and iron were determined colorimetrically by the phosphomolybdenum blue, permanganate, and o-phenanthroline methods, respectively. These chemical values were compared with spectrographic analyses of the same samples as obtained by the condensed direct current arc method. The comparative summary of spectrographic and chemical analyses plus the standard deviations of spectrographic values from chemical values is given in Table I. Standard deviations are calculated by means of the follon-ing mathematical expression:
where d
deviations of individual spectrographic results from chemical results n = number of comparisons =
For the total of 48 standard deviations calculated, in only 11 instances is the standard deviation larger than 10% of the chemical mean for the species and in no cases does it exceed 20y0. Copper was also determined spectrographically but no chemical analyses were available for comparison. To determine some measure of the reliability of the copper results as obtained by direct current condensed arc excitations, alternate copper concentration curves were constructed by the method of additions (1) whereby known increments of copper are added to a composite species mixture, followed by determination of copper to cobalt intensity ratios for each increment level. The analysis results for copper as determined from the established standard concentration curve and from the curve prepared by the method of additions were in very close agreement. Reproducibility. Ten aliquots of a sample of red clover ash were independently carried through the entire procedure beginning with solution of the ash in the internal standardhydrochloric acid solution. The statistical details obtained from the re-
producibility test are given in Table
11. SUMMARY
The method based upon direct current condensed arc excitation of acidic ash solutions as applied to spectrographic analysis of plant tissue is judged to be of higher precision than the copper electrode method, particularly for the analysis of the major ash elements, without sacrifice of any of the convenience generally associated with the copper spark technique. The procedure may be easily adapted for routine analysis of large numbers of samples. Purification of reagents is kept a t a minimum and chemical pretreatment of plant ash is confined to solution in an aqueous medium. The preparation of electrodes is easily and rapidly accomplished and the exposure time is short. The photometric procedure is based upon one internal standard line and one calibration curve for the determination of intensity ratios of the analytical lines, resulting in a corresponding simplification of photometric details. The difficulty encountered in alternating current spark excitation regarding individual emission characteristics of ash solutions derived from different plant species has been largely overcome with the use of direct current condensed arc excitation. The accuracy obtained over a range of seven species of plant tissues is well within spectrographic limits and the collective experimental studies indicate that this method is capable of an accuracy and a reproducibility which is superior to any method tested in this laboratory. LITERATURE CITED
L. H., “Spectrochemical Analysis,” p. 135, Addison-Wesley Press, Cambridge, Mass., 1950. Feldman, C., ANAL. CHEX. 21, 1041 (1949). Fred, M., Nachtrieb, N. H., Tonikins, F. S., J . Opt. Sac. Am. 37, 279 (1947). hlacDougal1, D., Biggs, D., ANAL. CHEX 24, 556 (1952). Mathis, JV. T., Ibid.,2 5 , 943 (1953). Young, H. Y., Gill, P. F., Ibid., 23, 751 (1951).
(1) Ahrens,
(2)
(3)
(4) (5) (6)
RECEIVED for review August 5 , 1957. -4ccepted January 11,1958. VOL. 30, NO. 6, JUNE 1958
1079
