fore, no antimony removal step was required. However, the presence of 200 y of antimony(II1) in the solution electrolyzed caused an error of 4%. A polarogram of ruthenium(1V) in IN sulfuric acid indicates that the reduction potential of ruthenium(1V) is -0.85 volt; hence, ruthenium(1V) will not interfere in the determination of uranium(V1). A series of 10 determinations of uranium(V1) made with 200 y of ruthenium(1V) present in the solution electrolyzed showed no interference. A solution of ruthenium(VI1) and (VIII) was prepared from a solution of ruthenium(1V) by oxidation with periodic acid. Five dcteriiiinations of uranium(V1) in the presence of rutheniuni(VI1) and (VIII) showed no deviation from the known uranium value. The low reduction potential of ruthenium(VII1) indicated that it is reduced immediately t o ruthenium(1V) in the presence of mercury at zero applied potential. A list of additional fission product elements that were checked for interference in the controlled-potential coulcmetric determination of uranium(V1) is given in Table 11. After approximately 1070 burnup of the fuel, the
minor fission product elements-i.e., those that have less than 1% fission yield-were calculated to be chemically insignificant. Cerium(1V) in 1N sulfuric acid has an oxidation potential of +1.40 volts and is capable of oxidizing ursnium(1V) to uranium(V1) ; this oxidation mould give a high value for the reductometric titration of uranium(V1). However, cerium(1V) is readily reduced t o cerium(II1) at zero applied potential and causes no inter. ference with the determination of uranium(V1). Polarograms of neodymium and ytterbium, representative rare earths, indicate that the rare earths are reduced at very negative potentials (more negative than - 1.0 volt) in 1N sulfuric acid and do not interfere in this method. Barium sulfate and some rare earth sulfates are removed during cleanup operations on the homogeneous reactor fuel. As a final check, five determinations of uranium(V1) were made on test solutions that contained all the fission product elements listed in Table I1 and the corrosion products listed in Table I in amounts designated in those tables. The recovery of the uranium known to have been present was 99.6%;
the relative standard deviation of the results mas 1%. ACKNOWLEDGMENT
The authors wish to thank H. C. Jones, Analytical Instrumentation Group, who constructed the coulometer; C. F. Leitten, Solid State Division, who furnished the europium oxide; and R. H. Busey, Chemistry Division, who furnished a pure solution of potassium pertechnetate. LITERATURE CITED
(1) Booman, G. L., ANAL.CHEM.29, 213 (195T’I. , \ -
~
(2) Booman, G. L., Holbrook, W. B., Rein, J. E., Ibid., 29, 219 (1957). (3) Carson, W.N., Ibid., 25, 466 (1953). (4) Carson, W. N:, personal communication. (5) Friedlander, G., Kennedy, L. W., “Nuclear and Radiochemistry,” rev. ed., pp. 74-6, Wiley, New York, 1955. (6) Furman, N. H., Bricker, C. E., Dilts, R. V., ANAL.CHEM.25,.482 (1953). (7) Kolthoff, I. M., Lingane, L. L., “Polarography,” Vol. 11,2nd ed., p. 436, Interscience, Yew York, 1952. (8) Ibid., pp. 546, 548. RECEIVEDfor review January 16, 1958. ilccepted May 16, 1958.
A Direct Flame Photometric Determination of Boron in Organic Compounds BRUCE E. BUELL Research Department, Union Oil Co. of California, Brea, Calif.
p A rapid, flame spectrophotometric method has been developed for determining boron directly in organic compounds. The method eliminates extractions or distillations of boron usually required b y chemical procedures and is suitable for boron Accuracy contents as low as 0.1%. and precision are on the order of 1 to 2y0of the amount present.
atomizing organic solutions directly into the flame. Numerous workers have applied flame photometry to various determinations directly in organic solutions (2-6, 7-12). The technique presented for boron determination is similar to that developed by Conrad and Johnson ( 2 ) for barium and calcium in lubricating oils. APPARATUS A N D REAGENTS
instrumentation has been applied to the flame photometric determination of boron by Bricker, Dippel, and Furman ( I ) and by Dean and Thompson (5). The latter authors reviewed determinations of boron by flame photometry and used acetone and alcohol mixed with water as solvents to enhance boron emission in inorganic compounds. The purpose of this investigation was to extend this application to the analysis of organic boron compounds and their concentrates in lubricating oils by ODERN
1514
ANALYTICAL CHEMISTRY
A Beckman Model D U spectrophotometer was used with the following Beckman accessories: a 9200 flame attachment, a 4300 photomultiplier, a 92300 spectral energy recording adapter, and a 4020 oxy-hydrogen atomizerburner. A Leeds &. Northrup Speedomax Type G recorder with a 10-mv. range and a chart drive of 2 inches per minute was used to record all measurements. Solvent mixture A was prepared by mixing equal volumes of 2-propanol and cleaners, naphtha. Solvent mixture B was prepared by
diluting 50 grams of 300 neutral oil to 500 ml. with solvent A. Boron standard solution, 400 mg. per liter. This solution was prepared by gently warming 0.571 gram of analytical reagent grade boric acid in 100 ml. of technical grade 2-propanol under a reflux condenser. When cool, the solution \vas transferred to a 250-ml. volumetric flask and 100 nil. of technical grade cleaners’ naphtha and 25 grams of neutral oil were added, The mixture was diluted to the mark with solvent A. All other calibration standards were prepared by quantitatively diluting this standard solution with solvent B. Sodium standard solution, 1000 mg. per liter. This solution was prepared by diluting 8.62 grams of Petronate H sodium sulfonate (2.9% sodium, L. Sonneborn Sons, Inc., New York, N. Y.) and 16.4 grams of neutral oil to 250 ml. with solvent A. All other sodium standards were prepared by diluting this concentrate with solvent B. INSTRUMENT SETTINGS A N D ADJUSTMENTS
The instrument settings used for
and 600 mM, re-
tivity ratios found with respect to water were 3.3, 10, and 13, respectively. Instrument sensitivity was unchanged, but the hydrogen pressure and mirror were adjusted to obtain maximum intensity in each solvent. The enhancement provided by 1 to 1 acetonewater is less than that obtained by Dean and Thompson (6).
0.020 mm. 16 p.s.i., varies for
MEASUREMENT OF BORON CONCENTRATION
measuring boron emission were a s follows: Photomultiplier voltage and recorder sensitivity
Adjusted to give a net emission of 80 for 400 mg. per liter boron standard and 1000 mg. per liter sodium
standard at 519.5
Slit Oxygen Hydrogen
ecr
E !
60’
E D
spectively
different burners 4 p.s.i., adjusted t o obtain maximum emission
To obtain maximum sensitivity and stability, the oxygen and hydrogen flow rates and the mirror in the burner housing must be adjusted. Adjustments, which depend on the element being determined and the solvent used, often have been neglected or improperly made. The following proccdures have been suitable when using oxygenhydrogen flames, but they are not entirely applicable to oxygen-acetylene flames. The height of the flame must be adjusted to obtain maximum emission while atomizing sample solution into the flame. Optimum hydrogen pressure for boron in 1 to 1 cleaners’ naphtha2-propanol is that which produces an oxygen-hydrogen flame, omitting sample solution, a little over 1 inch high. This hydrogen pressure is about one third of that required for aqueous solutions. For convenience, sodium in water is used as a reference standard for pressure and mirror adjustments. The flame height can be adjusted with either the pressure regulator or the screwdriver needle valve on the side of the control panel of the Beckman flame attachment. It will be necessary to adjust the screwdriver needle valve if the pressure giving correct flame hcight becomes too low for good control. It is preferable t o use a t least 4 p.s.i. The oxygen pressure should be adjusted to give maximum sensitivity. This will vary for different burners and depends mainly on the rate of sample atomization. Figure 1 shows the response of a typical burner to varying oxygen pressure, with the hydrogen pressure readjusted to give maximum boron emission for each oxygen pressure. An oxygen pressure of 16 p.s.i. was arbitrarily chosen as the minimum operating pressure giving essentially maximum sensitivity. This setting is not as critical as the optimum hydrogen pressure. The mirror in the burner housing must be adjusted. All mirror adjustments are made by trial and error movement of the lower mirror screw, after which the middle mirror screw must always be adjusted to obtain maximum emission. The upper mirror screw need not be adjusted. The
c
I
I
I
20
10
30
OXYGEN PRESSURE, P S I
Figure 1. Response of burner to oxygen pressure
atomizer-
Arrow indicates operating pressure chosen
position of the mirror is established by recording the position of the lower screw, For boron in solvent, the position of the lower screw for the final mirror adjustment is about three complete turns further out (counterclockwise) than for sodium in water. The final setting for boron in water is about one and one-half turns further out than for sodium in water. These instrument adjustments should be repeated until there is no further change, especially if large changes have occurred. CHARACTERISTICS
OF BORON SOLUTIONS
The most sensitive emission peak of the boron flame spectrum is centered a t 518 mp (Figure 2, A ) . The narrow peak a t 589 mp is due to a sodium impurity. The flame spectra of organic solvents contribute a n interfering emission, as shown by the spectrum of cleaners’ naphtha reproduced in Figure 2, B. Because this peak drops sharply to a level background at about 519 mp, a measurement of boron emission can be made a t 519.5 mp with no interference and little sacrifice in sensitivity. The solvent chosen for use in the procedure is a 1 to 1 mixture of cleaners’ naphtha-2-propanol, The flame background obtained with this solvent (Figure 3, D) is less than that obtained with cleaners’ naphtha alone. Conrad and Johnson ( 2 ) used 1 to 1 benzene-2-propanol, but this produces a less stable flame. Cleaners’ naphtha2-propanol mixture was chosen because of its good solvent properties and behavior in the flame. Other solvents probably could be found that are as good or better. This point was not investigated further except to compare the sensitivities for boron in 1 to 1 acetone-water, 1 to 1 cleaners’ naphtha2-propanol, and cleaners’ naphtha with the sensitivity in water. The sensi-
The relative emission of boron a t 519.5 mp, after subtraction of the solvent background, is proportional to the concentration of boron in the solvent. Measurement of unkn0n.n concentrations of boron would be very simple except that interference from sodium background emission is sometimes encountered. No other interference was observed. To correct for this interference, sodium background emission was calibrated at two wave lengths, 519.5 and 600 mp. Better sensitivity was obtained using this method rather than the technique of plotting differences employed by Dean and Thompson (6). A typical sodium flame spectrogram is shoFvn in Figure 3, C. Calibration of the background a t 600 mp revealed that the relative emission is approximately proportional to the sodium concentration. Furthermore, the relative emissions of sodium a t 519.5 mp and of boron a t 600 mp are proportional to the amounts of these elements present. The relationships of the relative emissions of these mutually interfering elements may be expressed by the two simultaneous equations Z5is.j
=
1600 =
+
aii[Bl a12 [ S al ~ i [ B l az[Sa]
+
where I = the relative emission, after subtracting the solvent blank, a t the wave length indicated by the subscript; a = coefficients: and [ ] = concentration of the element indicated. The solution of the two simultaneous equations for the concentration of boron is
If the relative emissions of the two elements are proportional to their concentrations, the values for a become constant and the solution may be reduced to [Bl
= kJug.5 - h.?Zsoo
The value of a in each case is the slope of the corresponding calibration curve which every laboratory must prepare from its own measurements. The values of k1 and k z are easily calculated from the respective value of a. The values obtained in the author’s laboratory are kl = 5.11 and k z = 1.38. If the relative emissions of the two elements in all cases are not proportional VOL. 30, NO. 9, SEPTEMBER 1958
1515
loor
I
I 460
440
I
I
480
500
WAVE
Figure 2.
1 520
r 55.3
L E N G T H , my
b.
20
I
0
Spectral curve of boron emission
Arrows at 519.5 and 600 mp indicate paints of measurement A. Boron standard, 400 mg. per lifer in 1 to 1 c l e a n e d naphtha-2propanol 3. Cleaners' naphtha
t o their concentrations, then values for a are functions of concentration and the simple solution given cannot be used. I n this case, the easiest method is a graphical solution by successive approximation. The wave length of 600 mp was chosen because the slope of the relative emission us. concentration plot for sodiiim is more nearly constant a t this xave length than a t 589 mp, the
Table I.
440
Figure
Determination of Boron in Organic Compounds,
3.29
Triisopropyl borate
5.75
5.73
Tri-o-cresyl borate
3.26
3.28
Tri-n-butyl borate
4.70
4.68
Tri-m-amyl borate
3.97
LENGTH. mp
1 520
J
j I
I
550
6LO
640
70
Flame Photometry -4v. Std. dev.
which is glass-stoppered. To bring all samples to approximately the same viscosity, the final solution is adjusted to contain 1 gram of oil per 10 ml. Thus, the sample size cannot exceed 2.5 grams. Multiply the sample weight by 10, dilute to that number of milliliters with solvent A, and then dilute to 25 ml. with solvent B. Thoroughly shake the samples, and determine the boron emission a t 519.5 mp. Verify that the flame photometer is maintaining calibration by frequent readings on the boron standard. If significant sodium is observed visually in the flame, also measure sample emission a t 600 m,u using the sodium standard to verify calibration. Calculate the boron content using values obtained from the calibration curres.
0.009
5.80
0.00
3.20
0.213
DISCUSSION
4.74
0.019
4.01
3.98
4 02
0.034
3.99 4.03 4.05 3 50 3.45 3.10 3 39 2 76
3.44
0.053
2.73
0.039
5.82
0.034
To establish precision and accuracy, a series of pure organic compounds was analyzed (Table I). Assay values s u p plied by manufacturers deviated no more than 1% of the amount present from the theoretical values, except for trimethyl borate which assayed a t 98.4% purity. Duplicate deterniinations were repeated on t\To different days for all except the first three samples. The precision attained was remarkably good for a flame photometric method. Comparison to the assay values reveals no bias. A very careful control in preparing the standard and adjusting the instrument to the standard was required for this degree of accuracy. Some difficulty was encountered at first, because of loss of volatile boron, but refluxing techniques for preparing the standard eliminated this.
3.45
Tri-n-octyl borate
2.72
2 74
2 7.5
Trimethyl borate
ANALYTICAL CHEMISTRY
WAVE
I
3.31
3.44
15 16
500
3.31 3.30 5.80 5.80 3.32 3.08 4.72 4.37 4.76 4 71
Tri-n-hesyl borate
Tri-(hesylene glycol) biborate 5.85
I 480
Arrows at 519.5 and 600 m p indicate paints of measurement C. Sodium standard, 1000 mg. per liter in 1 to 1 cleaners' naphtha-2propanol D. Blank, 1 to 1 cleaners' naphtha-2-propanol
Keigh a sample d m a t e d to contain 10 nig. of boron into a 25-m1. graduate,
...
I 460
3. Spectra! curve for sodium emission
PROCEDURE
Theoretical
q
----.D
I
' I 420
intensity is lox enough so that major readjustments of the instrument are not required for measurement, and background contributed by boron is low. This method has been applied to similar problems (IS).
Chem. Assay
Compound, Tri-m,p-cresyl borate
-
5.8G
10.3 10.3 5.81 5 87 5.81 5.84 5.79
As a further check on the precision of the method for application to determinations in oil, two blends in oil were prepared to represent a n average sample and a sample near the lower concentration limit of the method. Results are presented in Table 11. The method is applicable to any type of sample which can be dissolved in 1 to 1 cleaners’ naphtha-%propanol without markedly changing the viscosity from that obtained with a ratio of 1 gram of SAE 40 oil per 10 ml. of solvent mixture. The method was designed for determining boron in samples containing no less than 0.1%. It is possible to lower this limit by increasing the slit width and/or photomultiplier sensitivity. The limit of determination can be extended to 20 p.p.m., provided standards and samples are similar in composition and viscosity. The viscosity of the h a 1 solution must be low enough to allow uniform automization. ACKNOWLEDGMENT
Thanks are due to the U. S. Borax
Table
II.
Sample Boron Blend 1 0.0914
Blend 2
( 2 ) Conrad, -4. L., Johnson, IT. ANAL.CHERI.22, 1530 (1950). (3) Curtis, G. R.,Knauer, H.
Determination of Boron in Oil Samples
0.0887 0,0937 0.0932 0.456 0.450 0,459 0,454
Iv.
E., Hunter, L. E., Am. SOC.Testing Materials, Spec. Tech. Publ. 116, 67-74
(1951). (4) Curtis, R. E., Scott, R. W., Southwest Regional Meeting, .4CS, December 1954. (5) Dean, J. A., Lady, J. H., ANAL. CHERI.27, 1533 (1955). (6) Dean, J. A., Thompson, C., Ibid., 27, 42 (1955). (7) Fink, A., Mikrochim. Acta 1955, 314. (8) Gilbert, P. T., Jr., Am. Soc. Testing Materials, Spec. Tech. Publ. 116, 77-90 (1951). (9) Kingsley, G. R., Schaffert, R. R., J . Bid. Chem. 206,807 (1954). (10) Lady, J. H., dissertation, University of Tennessee, Knoxville, Tenn., 1955.
Std. Dev.
0.0918
0.0024
0.455
0 0044
C.,
and Chemical Corp. and the American Potash and Chemical Corp. for supplying the pure boric acid esters analyzed. Thanks are also due t o J. K. Fog0 for his aid in formulating the calculation method presented.
(11) hIalm, I., Herbert, F. J., Abstract
of Papers, 19P, 127th Meeting, ACS, April 1955. (12) Mohberg, M. L., Kaithman, T’. B., Ellis, W. H., Dubois, H. D., A4m. Soc. Testing Materials, Spec. Tech. Publ. 116,92-4 (1951). (13) Pinta, M., J . recherches centre natl. recherche sci., Lab. Bellevue (Paris) No.
LITERATURE CITED
21, 267 (1952).
C. E. Dippel, W. A,, Furman, X. H., d.S. Atomic Energy Comm. ReBt. H.Y. 0. 794 (1951) : S u clear Sn’. Abst. 6, 212 (1952).
(1) Bricker,
RECEIVEDfor review August 10, 1957. Accepted April 14, 1958.
Determination of Corticosterone and 17-Hydroxycorticosterone in Human Plasma JOSEPH McLAUGHLlN, Jr., THADDEUS J. KANIECKI, and IRVING GRAY’ Department of Chemisfry, Walter Reed Army Institute of Research, Washington 12, D.
C.
b A method for the qualitative and quantitative determination of corticosterone (CS) and 17-hydroxycorticosterone ( 1 7-OH-CS) in human plasma i s described. After extraction of the plasma with chloroform, the latter is removed, and the dried chloroform extract is chromatographed through a silica gel column. Depending upon the amount of ethyl alcohol in the chloroform, various steroids are eluted from the column and directly extracted with concentrated sulfuric acid. The sulfuric acid-induced fluorescences of the steroids are read on a Farrand fluorometer. Data obtained with the steroid standards and column blank enable calculation of the fluorescence produced per microgram of steroid and the percentage of separation of CS and 17- OH-CS in the ethyl alcoholchloroform fractions. Simultaneous equations may b e set up to solve for the concentrations of CS and 17-OHCS in the human plasma samples.
use of blue-tetrazolium (28)’ isotope dilution ( I , 17), and combinations of these techniques (1, 2, I d ) . With Sweat’s method (23-25) CS and 17OH-CS can be determined in human blood samples. The limitations of this method have bcen given by Weichselbaum and hIargraf (88) and Takeda (26). Takeda criticizes the Sneat method for the estimation of cortical hormones in plasma. He agrees that the 17-OH-CS is essentially correct, but believes that the CS-like substance contains material other than CS. The various methods have been discussed by Gold ( 9 ) . The method described in thiq paper is similar to Sweat’s (23-25), which is based on separation of CS and 17OH-CS by column chromatography, followed by sulfuric acid-induced fluorescence as described by Reichstein and Shoppee (2O), Wntersteiner and Pffiffner (SO), Peterson ( l i ) , Sweat (23-25), and Goldzieher (10). Of the corticosteroids likely to be present in
D
of corticosteroids in blood or plasma requires methods capable of measuring a fraction of a ETERMINATION
microgram. The first methods developd were bioassays such as Vogt’s (27) and Paschkis’ ( I O ) . Corcoran and Page ( S ) a c r e among the first to describe a chemical method for the determination of corticosteroids in plasma. Various other methods have been reported (3,,$,7 , 11, I S , 16, 19, 81,28, 29); those b y Porter and Silber ( I S , 19) and Xelson and Samuels (15) are considered the most practical for the routine determination of 17-hydrox-corticoids in blood or plasma. Most of the methods have been designed for the deterniinatioii of hydrocortisone (17-OH-CS) or total 17hydroxycorticoids and have neglected or ignored the presence of corticosterone (CS). CS is of special interest when studies involve experimental animals, because many species produce considerable quantities of CS. It is also important to know the CS concentrations in human blood samples. Both 17-OH-CS and CS may be determined by paper chromatography (1, 2, 6, 6, 12), polarography (14), a combination of the Porter-Silber procedure and the Mader-Buck method for
Present address, Quartermaster Research Command, Xatick, Mass. VOL. 30, NO. 9, SEPTEMBER 1958
1517
