V O L U M E 24, NO. 10, O C T O B E R 1 9 5 2 The foregoing assay procedure can be used to evaluate the content of desoxycholic acid in the presence of any of the above hy-
1667 The proposal of a reaction mechanism for the preceding chromophore formation would, a t this time, be speculative. Hoaever, observations made during the course of this study suggest the involvement of oxygen units with two unshared electron pairs. In the case of desoxycholic acid (3,12-dihydroxycholanic acid) and cholic acid (3,7,12-trihydroxycholanicacid) specific chromophores are realized with the prescribed vanillin reagent. Certain other sterols form specific chromophores, and for such sterols esterification does not change the quality of spectrum. Short-chain alcohols, low molecular weight acids, and water, when present in appreciable concentration, inhibit the chromophore formation described herein. If added subsequent t o the color development they quench the chromophore formation, and a negative rate curve will be observed. For this reason these substances are minimized in the reaction mixture. Lithocholic acid (3-hydroxycholanic acid) does not form a colored complex with the vanillin reagent Whether it combines in any way with the reagent is not known. I t s failure to form a chromophore makes possible highly specific assay procedure for desoxycholic acid especially when this acid is derived synthetically from cholic acid. LITERATURE CITED
450 500 550 WAVE LENGTH, rnr
Figure 4.
600
Spectra of Cholic Acid
droxy- and ketocholanic acids. The quantities of cholic and desoxycholic acids are evaluated simultaneously; no color is given with the ketocholanic acids and lithocholic acid.
Abe, Y., J . Bzochem. ( J a p a n ) ,25, 181-9 (1937). I b i d . , 26, 323-6 (1937). Chabrol, E., Charonnat, R., Cottet, J., and Blonde, P., Compt. rend. soc. b i d . , 115, 834-5 (1934). (4) Charonnat, R., and Gauthier, B., Compt. rend., 223, 1009-11 (1946). ( 5 ) Kaairo, K., and Shimada, T., 2. physiol. Ciiem., 254, 57 66 (1938). (6) Saalkowski, C. R., and hIader, TT’. J., d a a ~ .CHEM., 24, 1602 (1952). RECEIVED for review March 18, 1952.
Accepted July 2, 1952.
Application of line-Width Method of Spectrogram Evaluation to Spectrochemical Analysis of Plant Products ROBERT T. O’CONNOR AND D. C . HEINZEL3IAN Southern Regional Research Laboratory, New Orleans, La.
S E of the main obstacles to the extended use of spectrochemical methods for the quantitative determination of metallic cations in plant products has been the requirement of a large number of working curves. Plants and their products are submitted for analysis in an almost endless variety of widely diff went physical forms. The preliminary problem of reducing these samples to a form suitable for spectrochemical analysis has retarded widespread acceptance of this method in the analysis of plant products, in contrast to its extensive use in the analysis of metals and alloys where often a rod of predescribed diameter and length can be tooled and compared directly with readily available rods of standard composition. Generally applicable procedures for preparation of samples and a reduction in the requirement of working curves are essential if advantage of the simplicity and speed of spectrochemical methods for the analysis of plant products for metallic cations is t o be realized. A generally applicable method has been devised and evaluated for the ready analysis of plant products. It involves the preparation of ashes sufficiently buffered t o permit the evaluation of metallic cations in most plant products from a single set of working curves. A modification ( I d ) of the line-width method is used for the evaluation of the spectrograms. The line-width method was first thoroughly investigated by Coheur ( 4 , 6 ) , following simultaneous suggestions regarding its use by Eisenlohr and .4lexy (7) and by Gerlach and RollRTagen (9). The advantages of the method, arising from the fact that it is in-
sensitive t o time of exposure variations and t o changes in dcveloping conditions, and that it can be used ovsr wide ranges of concentrations, as it is independent of plate density and even not affected by self-absorption, have been pointed out by Ahrens ( I ) , Harvey ( 8 ) , and Nachtrieb ( I O ) . In spite of these advantages the line-width method does not seem t o have been generally adopted. A special application of the method t o the determination of relatively high concentrations of zinc (up to 10%) by Eastmond and JVilliams ( 6 )appears to be the only description of its use except in a paper from this laboratory (fa). This nonacceptance has been due, in part a t least, to difficulties in obtaining accurate measurements of line width. Coheur ( 5 )measured the width of the line of the internal standard a t a galvanometer reading equal t o the densest portion of the analysis line with a direct-reading Zeiss photometer or a modified direct-reading ?Jo11 photometer. Eastmond and Williams (6) describe their technique as follows: “For width measurements, line contours were recorded on photographic plates with the 16 mm. microscope objectives, a 0.5 mm. slit and a 6 4 X magnification on the recorder. The widths of the zinc line a t maximum density of the same barium and cadmium lines were measured from the photographic recording by means of a small scalemagnifier.” Use of an automatic recording microphotometer, Euch as the Leeds and Northrup instrument employed by the authors, and measurement of the line width directly from the profiles on the recorder strip, does not seem to have been de-
1668
ANALYTICAL CHEMISTRY
vribed. This modification has both simplified and increased the arcuracy of this measurement. JVith its use the line-width method of measurement has proved very satisfactory in reprodueibility and is advantageous as it permits measurements over a \vide range of concentration.. PREPARATION OF SAMPLES
As all plant and animal products are organic, ashing is an essential preliminary step and affords a means of reducing all samples to a similar form. I t has the additional advantage of conrentrating the metallic cations and greatly increasing the sensitivity of the analysis. Solut,ion methods are generally not witable as very few of the samples are completelj- soluble in common reagents. K e t ashing, involving use of mineral acids, was investigated and found unsatisfactory. D r y ashing proved most' satisfactory but must be conducted with a maximum of precaution (11). Tv-o procedures are offered. Selection of one or the other depends on the ash content of the sample analyzed.
80
-
I
I
I
I
I
Table
[.
Summary of Information for Preparation and Evaluation of Spectrograms
Spectrograph Slit Electrodes
Sample size Arc gap Excitation Region photographed Photographic plate Rotating sector Processing
Exposure time Spectral lines used as standards Spectral lines used for analysis Densitometer
5 mg. hlaintained a t 5 to 6 mm. D.c. arc, 230 volts, 25 amperes. Current regulated by resistance in series with arc 2300 t o 3300 -1. Eastman S A . KO.1 To reduce blackening in steps of 11.23 angular degrees Eastman Kodak D-19 developer and Easttuan Kodak fixer, formula h-o. F-5a. used with controlled temperature and tnechaniral agitation 90 seoonds to assure complete burning. S o preburn h l g : 2942.11 hfg I. 3336.68 hfg I Ge: 3269.49 Ge I Ge: 3039.06 Ge I Cu: 2961.16 Cu I , 3273.96 Cu I Fe: 3017.63 Fe I, 3020.64 Fe I. 3057.45 Fe I h l n : 2576.10 M n 11.2949.20 Mn. 3054.36 M n Automatic rerording Leeds a n d S o r t h r u p microphotometer
I
ash is finely ground and thoroughly mixed in final preparation for analysis. Working standards are obtained by ashing, in identical manner, portions of the magnesium nitrate solution containing graduated amounts of the various elements to be determined. Working curves for iron over a concentration range of from 3 parts per million to about 1% obtained by measurement of the profiles of the spectrograms are shomn in Figure 1, A . Procedure B. Ash Content of Sample over 1%. Exactly 5 grams of sample are ashed in a tared Vycor dish as described in Procedure A, but without addition of the magnesium nitrate solution, After removal from the furnace the dish is cooled and reweighed to determine the ash content of the sample. A volume of a very dilute aqueous solution of germanium dioxide is added so as to introduce a quantity of the dioxide equal to 4.14% of the ash. The ash when dried, ground, and thoroughly m k e d is ready for analvsis. LOG. CONCENTRATION
Figure 1.
P. P.M.
Working (or Analytical) Curves
Determination of iron by Procedure A for plant product of low ash content Fe 3020.64 A. 2. Fe 3057.45 A . 3. Fe 3017.63 A . H . Determination of copper b y Procedure B for plant product of high ash content I . Cu 3273.96 A., sector set to admit 18.8% of light 2. Cu 3273.96 A., sector set t o admit 6.3% of light
A.
G e 3209 4 9 A
1.
'q Exactly Procedure A. A s h Content of Sample Less Than 1. 16.67 grams of the sample are weighed into a Vycor dish (90-mm. diameter), and 0.50 gram of magnesium nitrate in ethanol ( 2 ml. of a solution of 250 grams of magnesium nitrate hexahydrate per liter of 95% ethanol) is added. The reagents used must be free of the trace metals for u-hich samples are to be analyzed. Obviously the ratio of sample weight to the magnesium nitrate buffer is arbitrary and can be varied for different samples depending upon the sensitivity required, if the ratio actually used is considered in obtaining and using the working curves. T h e ratio suggested here A ill permit analysis with a sensitivity of about 1 part of metal in 10,000,000parts of sample for the more sensitive metals, copper and iron, for most types of samples. Ashing with magnesium nitrate is, of course, well known ( 2 ) . The procedure was adapted from t h a t used in a micromethod for phosphorus (3) using ethanol instead of hydrochloric acid as a solvent. T h e dish is covered with a n inverted short-stemmed borosilicate glass funnel, with a maximum diameter less than the maximum diameter of the dish. The sample is heated on a hot plate, and the temperature is gradually and cautiously raised until a temperature of approximately 300" C. is attained. The charred sample is then ashed in a muffle furnace, with a n initial temperature of 225" C. increasing in increments of 25" C. a t 30minute intervals until a temperature of 450" C. is reached. T h e samples are held a t this temperature in the furnace overnight. They are then removed, cooled, and quantitatively transferred to a small mullite mortar with aid of a camel's-hair brush. The
C u 3 2 7 3 96 A
A-7 Figure 2. Reproduction of Portion of Itrcorcler Strip Chart Obtained for Spectrogram Evaltcatiori. Using Recording \Iicrophotometer A.
Determination of iron i n sample of l o w ash content b y Pro-
8.
Determination of copper i n sample of high ash content b y Procedure B
cedure A
Working curves are obtained by preparing a mixture of salts representing the major constituents of a typical ash. The particular mixture selected contains 6E1.187~potassium carbonate, 0.96% sodium chloride, 11.02% calcium carbonate, 18.70% magnesium carbonate, and 4.14% germanium dioxide. Such a mixture is believed to be sufficiently representative of the ash of most plants so as to avoid any significant error due to extraneous ion effects. T h e amount of germanium dioxide, selected as an internal standard, was obtained from study. of photographic densities, Under the conditions of photographing, thls quantity
V O L U M E 24, NO. 10, O C T O B E R 1 9 5 2 Table 11.
manganese contents of a variety of sample materials. It has also been used for Manganese the quantitative determination of boron, Arerage Deviation zinc: aluminum, nickel, and tin. found, from mean, p.p.m. 70 I n Table I11 some results of recovery 6.2 13.2 tests and the analysis of standards ai’e 12.0 18 8 given. Recovery tests were made by 32.0 9 0 298.8 10.3 adding known amounts of the specified 15.8 12.4 elements to additional portions of samples 4.1 7.9 previously analyzed, reashing, and coni0.4 9.1 pletely reanalyzing. Standards were pre0.0 .. 0 6 7.0 pared t o represent, as closely as pos0 . 0 . . sible, the compositions of the various 0 0 samples including the trace metals. These samples were then both ashed and analyzed in the same manner used for the actual sample?. Both recovery tests and analysis of standards were conducted b y Procedure -1 or Procedure I3, whichever was applicable-i.e., recovery tests were made in the same manner used for the original sample and standards were analyzed by Procedure A if the aph content was less than lyO,otherwise by Procedure B. I n some borderline case$ just about 1% both procedures n-ere where the ash content w a ~ w e d in these determinations. Satisfactory agreement is an indication that the bufferinn was adeauate in both cases. There data, as well as considerable additional data of the same nature, show that the accuracy of the method for a single determination n-ill be within 12075, usually within ?CIS%, of the amount of the element precrent in the sample. If the average of triplicate determinations is used, accuracy within i i to +8y0 for the elements can be attained for the lower concentrations, 1 part of element in 10,000,000 parts of sample, with somei\-liat better accuracy for samples containing the metallic cations in higher concentrations. The lower limit of sensitivity can b r increased by increasing the ratio of sample to magnesium nitrate buffer. The limiting factor occurs y h e n insufficient buffer is provided to permit highest quantitat,ive accuracy due to cxtraneous ion effects. The upper limit of the method can be extended alniost indefinitely by t,he selection of less sensitive lines when evaluating the spectrogram, or by use of the line-width method for higher concentrations as described by Coheur ( 4 ) and a s illustrated by Eastmond ( 6 ) in the determination of zinc-i.r., liy measuring- the v i d t h of a line of the element being determinrd :it the maximum density of a weak standard line.
Trace hIetal Content of Specified Plant Products Copper
Iron
Average Deviation found, from mean, p.p.m. 70
Average from Deviation foiind. mean, 70 p.p.m.
-~
Plant Product Cotton lint Cottonseed kernels Peanut kernels Rice bran Sweet potatoes Isolated cottonseed protein Crude cottonseed oil Hydrogenated cottonseed oil Crude peanut oil Commerpial G u m rosin shortening
Table 111.
1669
1.5 16 0 2?.? J
.J
14.2
104.9
