to explode violently if the water is not present. The use of ice water in the bath for Procedures D and E as a rate-controlling device is not recommended. It is believed there is some possibility that initiation of oxidation could be delayed until a considerable amount of H202 were present and that then some critical state of concentration and localized reaction could take place and cause detonation of the total mixture. A room temperature water bath appears to be adequate and advantageous. The procedures given have been evaluated on compounds containing the elements C, H,0, K,Br, P, and 13. It is noteworthy that for the one compound available containing phosphorus, P-hexamethyltriborophane. excellent results for boron content were obtained by Procedure D. The 41.93% phosphorus content of the sample gave no interference. Because phosphoric acid does interfere with the titration for boron, it may be concluded that treatment D did not convert the phosphorus to H3P04. The absence of H3P04 was also demonstrated by adding a solution of 13i(h’03)nto a solution of the P-hexamethyltriborophane resulting from treatment D. KO precipitate was formed. If phosphorus interference is encountered, the use of Bi(NO& for removal of the phosphorus in the manner
activity; Lohr A. Burkardt for encouragement in carrying on the project, and Ronald A. Henry and Wayne R. Carpenter for samples and counsel.
described by Kilson and Pellegrini ( I d ) or the application of anion exchange resins as described by Wolszon, Hayes, and Hill (IS) may give a satisfactory solution to the problem. I t is hoped that few, if any, samples will be encountered which cannot be analyzcd by the six-member series proposed. If some do appear it would be feasible ‘to expand the series by applying hydrogen peroxide oxidation in the absence of any water and possibly with a small amount of trifluoroacetic anhydride added to ensure dry conditions. I n other words, this suggested technique would be the Strahm and Hawthorne ( 2 1 ) method modified only to exclude acetonitrile and to use some trifluoroacetic acid as replacement for part of the trifluoroacetic anhydride originally specified. Oxidation in the oxygen bomb (Parr) might also be useful in developing procedures if compounds containing phosphorus or fluorine prove troublesome.
LITERATURE CITED
(1) Abbott, R. M., Liszt, N. M., Roth, Milton, Tech. Note FRL-TN-85, Picatinny Arsenal, Dover, K. J., September 1961. (2) Corner, M., Analyst 84, 41 (1959). (3) Cosgrove, J. D., Shears, E. C., Ibid., 85, 448 (1960). (4) Dunstan, Ivan, Griffiths, J. V., ANAL. CIIEY. 33, 1598 (1961). (5) Furman, K. H., ed., “Scott’s Standard Methods of Chemical Analysis,” Vol. 1, 5th ed., p. 176, Van Nostrand, New York, 1939. (6) hlartin, J. R., Hayes, J. R., AKAL. CHEM. 24, 182 (1952). (7) Shaheen, D. G., Braman, R. S.. Ibid., 33, 893 (1961). (8) Smith, T. B., “Analytical Processes,” 2nd ed., p. 207, Edward Arnold, London, 1940. (9) Snyder, H. R., Kuck, J. A., Johnson, J. R., J . Am. Chem. SOC.6 0 , 110 (1938). (10) Steyermark, Al, “Quantitative Organic Microanalysis,” The Blakiston Co., New York, 1951. (11) Strahm, R. D Hawthorne, h l . F., ANAL.CHEM.32,’530 (1960). (12) Wilson, H. pi., Pellegrini, G. U. M., Analyst 86, 517 (1961). (13) Wolszon, J. D., Hayes, J. R., Hill, R. H., ANAL. CHEM.29, 829 (1957). RECEIVEDfor review March 19, 1962. Accepted August 17, 1962. Analytical Division, 141st Meeting ACS, Washington, D. C., March 1962.
ACKNOWLEDGMENT
The author is indebted to R. Donald Strahm, Rohm and Haas Co., Redstone Arsenal, Huntsville, Ala., and to Frank Radovich, American Potash and Chemical Corp., Los Angeles, for supplying a number of the samples. The author is grateful t o the following persons at his
Determination of Parts per Billion Iron FIuorescence Extinction
by
JACOB BLOCK and EVAN MORGAN Olin Mafhieson Chemical Corp., New Haven, Conn.
b A fluorescence extinction method for determination of iron in the part-perbillion range has been developed. The method is based on the reaction between iron and the fluorescent aluminum-Pontachrome Blue Black R complex. The fluorescence intensity of the complex decreases sharply in the presence of iron, the decrease being linear over a concentration range of 0.02 to 0.2 pg. of iron per ml. of final solution. The sensitivity of the method is three times greater than the spectrophotometric 4,7-diphenyl- 1,lO-phenanthroline procedure.
T
arose for a method to determine iron with a sensitivity significantly greater than that available with spectrophotometric procedures. The high sensitivity of fluorometric procedures is well known, but a survey of HE NEED
the literature indicated that no fluorometric method for iron had as yet been developed. The present method utilizes the interference of iron in the fluorometric determination of aluminum with Pontachrome Blue Black R(PBBR) ( 2 ) . The fluorescence of the aluminum-PBBR complex is destroyed by traces of iron, and this decrease in fluorescence is a direct function of the iron concentration. The sensitivity of the method is three times greater than the 4,7-phenyl1,lO-phenanthroline procedure-i.e., 0.25 pg. of Fe/ml. will produce a 20% decrease in transmittance using 4,7diphenyl-I ,lo-phenanthroline (1), whereas in the present method 0.08 pg. of Fe/ml. will produce the equivalent decrease in intensity. EXPERIMENTAL
Except for the organic dyes, all chemicals were reagent grade, Reagents.
and all were used with no further purification. Pontachrome Blue Black R was obtained from E. I. du Pont de Nemours and Co., Wilmington, Del. (Superchrome Blue 13 Extra, Allied Chemical Corp., New York, is reportedly the same dye.) Superchrome Violet B was obtained from the Allied Chemical Corp. Aluminum-PBBR Solution. A solution [30.0 mg. of KAl(S04)2.12He0dissolved in a few milliliters of distilled water] was placed in a 100-ml. volumetric flask. Next, 12.5 grams of ammonium acetate were dissolved in a minimum of distilled water and added to the flask, followed by concentrated sulfuric acid until the solution cleared. Finally, 37.5 ml. of a 0.1% solution of PBBR in 95y0 ethanol were added, and the solution was brought to volume with distilled water. Standard Iron Solution. Mallinckrodt iron wire (68.5 mg., 99.9%) was VOL. 34, NO. 12, NOVEMBER 1962
1647
v)
301
,
so
100
200
150
250
300
3!
TIME (MIN.) I
2
4
3
5
Figure 2.
Fluorescence intensity as a function of time
A. 0.5 pg. Fe/25 ml.
1
I
I
I
2
I
I
I
3
4
5
B. 10 pg. Fe/25
ml.
p g . FeI25ml.
Figure 1 . Fluorescence intensity as a function of iron concentration A. 12.5 pg. AI, 4.5 hr. B. 12.5 pg. AI, 1 hr. C. 25 f i g . AI, 2.5 hr.
D. 25 pg. AI, 1 hr. C. 50 pg. AI, 1 hr. F. 50 pg. AI, 72 hr.
dissolved in dilute sulfuric acid and brought to volume in a 100-ml. volumetric flask. -4 5.0-ml. aliquot was taken and brought to volume in a 10-ml. volumetric flask, and a 3.0-ml. aliquot of this solution was brought t o volume in a 100-ml. volumetric flask. The final solution contained 10.3 pg. of Fe/nil. All other bolutions were prepared on ~a weight basis. ADDaratus. A Becknian Model DU spe&ophotometer equipped with a T o . 22850 fluorescence attachment was used for all fluorescence measurements. -1 Corning No. 9863 filter, which transmits between 240 and 400 nip, was used as the primary filter in conjunction with the mercury lamp. The Model DU was also used to determine absorption spectra. A Beckman ;\lode1 H2 pH meter was used for pH measurements. Procedure. The method can be used directly on solutions ivhich do not contain either great aiiiounts of metallic ions or organic material. Iron in organics can be determined by means of a preliminary ashing with a subsequent addition of a small quantity of HC1. (Large quantities of reagents, such as mineral acids, should be avoided as these contain appreciable quantities of iron.) Large 1648
ANALYTICAL CHEMISTRY
70
I
I
40
4.5
I
I
5.5
5.0
6.0
PH
Figure 3.
Fluorescence intensity as a function of p H A. N o iron,
quantities of metals inust be removed prior to the determination. Prepare a series of standards ranging from 0.5 p g . to 10 pg. of iron in a x olume not exceeding 15 nil. Bring all volumes to 15 nil. with distilled water, carrying 15 ml. of distilled water as a blank. Add by pipet 2 ml. of the aluminum-PBUR solution to each sample, and bring to p H of 4.8 + 0.1 with ammonium hydroxide or sulfuric acid. Heat t o near boiling on a hot plate, cool, and transfer each to a 25-ml. volumetric flask and bring to volume nith distilled water. Treat the sample in a similar manner. Calibrate the fluorometer nith the blank solution set to read 1 0 0 ~ otransmittance. using an ultraviolet excita-
6. 2 pg. Fe/25 ml.
tion source. Expose the solution to the ultraviolet beam only long enough to measure the transmittance. The fluorescence peak is nieasured a t a wavelength of 595 mb. Plot per cent transmittance us. micrograms of Fe and determine the amount of iron in the sample from the graph. RESULTS AND DISCUSSION
Fluorescence Intensity a s a Function of Iron Concentration. Figure 1 shows the T ariatioii of fluorescence intensity a s a function of iron concentration for several aluminum concentrations and a t several time intervals. The instrument scale was
Table I.
Effect of Heat Scale Time, reading, Sample" hr . %T 74.0 A 1 A 22 74.1 67.3 B 1 B 22 73.8 B, a t room a .4,heated to near boiling. temperature.
reset arbitrarily for each run, so t h a t readings are not necessarily transferable from one graph to another. Except for the addition of iron, the procedure described by Weissler and White ( 2 ) (scaled down to a final volume of 25 ml.) was followed. The results indicate that equilibrium is not quickly established, but once it has been attained, the decrease in fluorescence intensity is a direct function of the iron concentration. The line corresponding t o 25 pg. of A1 had the greatest slope, and this concentration of Al was chosen for further study. Fluorescence Intensity as a Function of Time. Figure 2 is a plot of the fluorescence intensity as a function of time for samples containing 0.5 pg. of Fe/25 ml. and 10 pg. of Fe/25 ml. Equilibrium is attained earlier for the 10-pg. sample. Effect of Heat. Two samples, each containing 2 pg. of Fe and aluminumPBBR reagent were prepared. One of the samples was heated to near boiling before being brought t o final volume. The results, summarized in Table I, show that heat will hasten the attainment of equilibrium without destroying the organic dye. Effect of pH. Little difference in sensitivity was found over the p H range 4.0 to 6.0 (Figure 3). Therefore, a p H of 4.8, the p H of maximum aluminum-PBBR fluorescence, was chosen for the method. Use of Aluminum-PBBR Reagent. A stock aluminum-PBBR solution was prepared so t h a t 2 nil. would be equivalent to the total quantities of reagents used in each of the above experiments. T h e mole ratio of aluminum t o dye was altered from approximately 1 to 2 to approviinately 1.3 t o 2 to minimize the effect of any aluminum nhich might be present in saniples t o be analyzed. (According to Keissler and White ( 2 ) , the fluorescence intensity remains fairly constant at aluminum-to-dye mole ratios greater than 1 t o 2.) Varying quantities of iron were added t o 2-ml. aliquots of the reagent, which were then brought to p H 4.8, heated to n m r boiling, cooled, and brought to volume in 25-ml. volunietric flasks. The fluorescent intensities were then measured, and a plot of yOT US.
Figure 4. Absorption spectra A. AI-PBBR
f 50 pg. Fe
8. PBBR f 50 pg. Fe
iron concentration yielded a straight line. The results indicate that the mixed reagent can be used for 5 pg. of iron or less, and that a longer heating period will probably be required for samples of higher concentration. Effect of Diverse Ions. T h e effect of diverse ions is summarized in Table 11. T h e results indicate, for
Table II. Effect of Diverse Ions Fe concentration, 1.0 pg.125 ml. Fe Rel. Diyerse fig.125 found, ion ml. error Pg. ~a41(III) 2.5 1.0 0 5 0.4 - 60 Co(I1) 1 1.1 10 la 3.0 200 5 4.2 320 Cu(I1) 1 0.9 - 10 5 3.2 220 Cr(II1) 5 1.0 0 Mn(I1)
10
1.0
3
1.0 1.2
Xi(I1)
5 3 5
Ti(1V)
1
V( V)
5 1
Zn(I1)
5 5
Zr(1V) F-
10 3 5 5 5
1.0
1.3 1.0 2.6 1.2
0 0
20 0 30 0
160 20
Table 111.
Effect of Ferrous Ion HydrpxylFe(III), Fe(II), amine Scale pg./25 pg./25 hydroreading, rnl. id. chloride 96 T 0
280
10
1.0 1.0
0 0
1.1 1.2 2.6
10 20 160
0 0 10 0 0 0 0 5
40
D. PBBR
the determination of I pg, of Fe, that at least 10 pg. of Cr(II1) and Zn(II), 3 pg. of Mn(II), S ( I I ) , Al(III), and 1 pg. of Ti(1V) and Zr(1V) do not interfere. Co(III), Cu(II), and V(V) show slight interference (10 relative per cent for 1 pg. of diverse ion), and most likely these can be tolerated a t lesser concentrations. Fluoride and phosphate interfere seriously if present. Effect of Ferrous Ion. Samples of the aluminum-PBBR reagent were run with varying amounts of Fe(II), Fe(III), and hydroxylamine hydrochloride. T h e results are tabulated in Table 111. One can conclude t h a t iron(II), in the absence of a reducing agent, is converted to iron(II1). Iron(II), stabilized by a reducing agent, decreased the fluorescence of the aluminum-PBBR complex. but t o a lesser extent than iron(II1). Therefore, the present method can determine
3.5
PO?-^ 1.4 a Superchrome Violet B.
C. AI-PBBR
5
0
0
0
0
10
10 0 0
0 5 5 0 0
0
1 drop of 1% 1 drop of lY0 1 dropof ly0
2 ml. of 100,
79,s :ax 2
39 2
45 8 46 3 79.1 80 8
0
60.2
0
69.8 60.3 70.8
2 ml. of 10% 2 nll. of 10%
VOL. 34, NO. 12, NOVEMBER 1962
1649
totalsiron as long as no agent capable of reducing iron(II1) is present. Reaction Mechanism. Absorption spectra were taken of various combinations of aluminum, iron, and PBBR with water as the blank (Figure 4). The two iron-containing samples absorb a t 400 mp, whereas PBBR and aluminum-PBBR absorb only dightly a t this wavelength. These measurements lead to the conclusion that iron destroys the aluminum-PBBR complex to form a stronger nonfluorescent complex with PBBR. Use of Superchrome Violet B. Weissler and White, in their method for aluminum, reported a greater interference from iron when Super-
chrome Violet B was used in place of PBBR. Consequently, a 0.1% solution of Superchrome Violet B in 9570 ethyl alcohol was prepared, and a standard curve for iron was determined. However, the sensitivity of the reagent was equal to the sensitivity obtained with PBBR. but the interference from cobalt was greater (Table 11), and therefore no further work was done along these lines. Stability. The fluorescent intensities were stable for a period of a t least 48 hours. Accuracy of the Method. NBS samples of nickel-silver (157a) reported to contain 0.174Oj, Fe were analyzed by the present method.
Copper and nickel, the major constituents in addition t o zinc, were first removed by electrolysis and precipitation as the dimethylglyoximate, respectively. The results obtained on two aliquots mere 0.176 and 0.171% Fe. LITERATURE CITED
(1) Diehl, H., Smith, G. F., “The Iron
Reagents.” G. F. Smith Chemical Co., Columbus, Ohio, 1960. (2) ~7eissler,A., White, C. E.. ANAL. CHEM.18, 530 (1946). RECEIVEDfor review June 22, 1962. Accepted August 31, 1962. Division of Analytical Chemistry, 142nd Meeting, ACS, Atlantic City, N. J., September 1962.
Semimicro and Micro Steam Distillation The Estimation of the Essential Oil Content of Small Plant Samples W. J. FRANKLIN and H. KEYZER Museum o f Applied Arts and Sciences, Sydney, New Soufh Wales, Australia
b Two micro steam distillation units have been designed to estimate the volatile oil content of small plant material samples. The results obtained are of an accuracy comparable to that of conventional macromethods, although the volumes of oil recovered amount to only a few microliters. In some instances a single leaf may constitute a sufficient sample. A semimicro steam distillation apparatus has also been developed for the same purpose. In addition, the three stills as a group can at least partially obviate the problems often encountered in the purification of small amounts of certain organic liquids.
T
HE MARKED INCREASE in the use of infrared spectroscopy and gas liquid chromatography in phytochemistry during the last decade has necessitated the development of suitable techniques for handling very small quantities of essential oil constituents. Furthermore, current botanical research into the genetics, nutritional requirements, and biosynthetic mechanisms of essential oil bearing plants necessitates the chemical investigation of very small amounts of plant tissue, involving the quantitative recovery of oil volumes ranging from 1 to 100 d. EXPERIMENTAL
Microdistillation units have previously been described ( 2 ) ; so also has a semimicro Apparatus and Procedure.
1650
ANALYTICAL CHEMISTRY
steam distillation assembly (2). However, none of these is suited to the needs described above. The specific need for a steam distillation technique arises from the known stability of some very reactive essential oil components to steam and the effective liberation of oil from the sacs in the plant tissue. Still 1. The apparatus show? in Figure 1 is termed a semimicrocohobation .till. The term “cohobation” implies circulative steam distillation, in which the distillate is condensed and allowed to separate into oil and water layers. the aqueous phase then being returned to the distillation vessel for further use. Still 1 is a modification of the IIcKern and Smith-White apparatiis (SI, but allows determination of oil quantities to a lower limit of 50 pl. The semimicroapparatus differs radically in the system of measurement. The leaf sample is weighed into a 250ml. flask containing sufficient water. The contents of the flask are boiled. The condensate enters C, and its momentum forces the oil into D, where the oil separates as the top layer while the water returns through the rubber tube F to the flask. On a 30-gram leaf sample, distillation is complete in 2.5 hours. A rubber stopper is inserted in C, the rubber tube F is clamped off, and stopcock E is closed. A hypodermic needle fixed to a separatory funnel G filled with saturated sodium chloride solution is inserted into the rubber tube F. Another needle, fitted to a graduated pipet I (size appropriate to the collected oil) is also inserted into F . On opening stopcock H , the pipet fills
with salt solution. Stopcock E is opened and the oil level in D is allowed to rise into the capillary to a convenient mark-for example, K-whereupon E and H are closed. The pipet is read and stopcock E is reopened. The oil is allowed to pass K until the bottom level reaches I
