Microwave spectroscopy analysis of the distribution of deuterium in

Chemical Analysis by Microwave Spectroscopy. Gordon E. Jones , Robert L. Cook , David R. Lide. C R C Critical Reviews in Analytical Chemistry 1973 3 (...
0 downloads 0 Views 764KB Size
Download PDF
atoms in different molecular environments in much the way that is common in X-ray PES (cf. the different sulfur-peaks in disulfoton and oxygen peaks in butonate). So far most of the compounds subject to UV-PES have had a high degree of symmetry, so this possibility has not been made evident. It is an exciting possibility, especially as the chemical shift in X-ray PES may be nullified if there are two opposing tendencies such as ligand to metal cr-donation and metal to ligand a-back donation (17). We also feel that the simple arguments used to explain the spectra could well be amplified (or vitiated) by a more detailed analysis of the spectra in which band areas are more carefully measured and correlated with ionization cross-sectional areas. A program of computer analysis of the spectra is in hand to carry out this work. As it stands, we feel this study shows that it may be possible to assign peaks in complex spectra by simple methods. Although the assignments may need revision or incompletely or inadequately reveal the true nature of the orbital from which ionization occurs, the methods proposed do allow the correct number of bands and their approximate order to be deduced. As before we note that the spectra are quite distinct and form the basis of identification, also that the peculiarly phos-

phorus characteristics of P lone-pair and the oxygen lone pair in the P=O moiety are clearly observable in the spectra. The possibilities for detecting trace impurities, such as water in PCI5, and of resolving the spectra of mixtures have been demonstrated. Quantitative application of the above findings will require further work on the inlet system so that the amount of sample entering the target chamber is known as well as better handling of the output data. Work on these matters is in progress, and preliminary results from a computer program fully confirm the hand calculations reported here (18).

(17) W. E. Morgan, W. J. Stec, R. G. Albridge, and J. R. Van Wazer, Inorg. Chem., 10, 926 (1971).

(18) D. Betteridge, M. A. Stevens, and M. Thompson, University College Swansea, unpublished work, 1971172.

ACKNOWLEDGMENT

Thanks are due to Albright and Wilson and to Shell for the provision of the various samples examined in this work. We are also grateful to Perkin-Elmer Ltd. (Beaconsfield) for the use of a model PS18 photoelectron spectrometer. RECEIVED for review February 11, 1972. Accepted June 5 , 1972. We gratefully acknowledge the support of the Agricultural Research Council, who provided the instrument and a Fellowship to A.D.B., and of the Science Research Council, who supplied a Fellowship to M.T. and a maintenance grant to N.R.K.

Microwave Spectroscopy Analysis of the Distribution of Deuterium in Propene-D, Obtained from Catalyzed Hydrogen-Deuterium Exchange Reactions LeRoy H. Scharpen and Roger F. Rauskolb Scientijc Instruments Division, Hewlett-Packard Company, 1601 California Auenue, Palo Alto, Calg. 94304

Chadwick A. Tolman Central Research Department, E . I . du Pont de Nemours & Company, Experimental Station, Wilmington, Del. 19898 Five subspecies of propene-d,, differing in the location of the deuterium atom, can be distinguished by microwave spectroscopy. Quantitative analysis of these subspecies by microwave spectroscopy was done for samples obtained by hydrogen-deuterium exchange between CHsOD and propene-do in the presence of homogeneous catalysts of Pt, Rh, and Ni. The relative amount of each subspecies in a sample varied considerably for the catalysts used. The relative standard deviation of the analytical result was about 2% of the values found for subspecies concentrations ranging from 1.5% to 17% of the total sample. Most spectrometer functions, including signal processing, were controlled by computer.

MICROWAVE SPECTROSCOPY is the study of the centimeter and millimeter wavelength absorption spectra of molecules in their vapor state at low pressure. Although this technique has been used to study a variety of chemical problems ( I ) , there has been a paucity of reported analytical applications (1) D. R. Lide, Jr., Surc. Progr. Chem., 5 , 9 5 (1969). 2010

(I, 2) and, as a result, a lack of knowledge among those interested in chemical analysis concerning the capabilities and methodology of the technique. Lide (I) recently listed the lack of reliable, easy to operate spectrometers as one reason for slow development of the analytical potential of the technique and problems associated with accurate measurement of absorption line intensities, required for quantitative analysis, as another. The availability of commercial spectrometers has obviated Lide’s first point. In this paper, we consider some aspects of the second question, the reliability and accuracy of quantitative data. To do this we treat our solution of a specific analytical problem-namely, the determination of the amounts of the five subspecies of propene-dl (C3HsD) present in samples of partially deuterated propene obtained in catalytic hydrogen-deuterium exchange reactions. Interpretation of the analytical data in terms of the mechanism

(2) L. H. Scharpen and V. W. Laurie, ANAL.CHEM., 44, 378R (1972).

ANALYTICAL CHEMISTRY, VOL. 44, NO. 12, OCTOBER 1972

C

/

C

\

HI

Figure 1. Numbering of hydrogen atom positions in propylene. Positions 4 and 4’ are out-of-planeand are equivalent governing the exchange reaction will be reported elsewhere (3). Our particular analytical problem arises as follows. In the presence of a suitable catalyst, olefins and deuteriumcontaining compounds such as C H 3 0 D exchange hydrogen and deuterium ( 4 , 5). For olefins containing structurally different carbon-hydrogen bonds, the exchange rate can differ with hydrogen site. The distribution of deuterium among the possible sites is determined by the exchange mechanism and the relative rate constants involved. Quantitative measurements of the deuterium distribution in reaction prodhcts, therefore, provide the kind of detailed data necessary for elucidating reaction mechanisms and assessing differences in the activity of catalysts. Microwave spectroscopy is particularly suited for such anaIyses. Changes in the absorption frequencies upon isotope labeling of a compound will generally be quite large (except for nuclei near the molecular center-of-mass) and will depend upon both the mass change and the position of the label nucleus in the molecule (6). For propene, five subspecies of the mono-deutero compound, formed by deuterium substitution at one of the numbered hydrogen positions in Figure 1, produce distinct microwave spectra (7). For convenience, we can refer to each mono-deutero species by the position of substitution. Thus, for example, the HI species is cis-CH3CHCHD. Two of the five species, H 4 (equivalent to H 4 f )and Hs, are rapidly interconverted by internal rotation of the methyl group about its axis and always occur in a 2 :1 ratio (7). Measurements on these two species provide a n internal check on the accuracy of the method from a comparison of the experimental and theoretical values. Hirota and coworkers (8-10) previously reported similar analyses of propene-dl samples and also found (9) that infrared absorption and nuclear magnetic resonance methods were insufficient for a detailed quantitative analysis. Our work (3) C. A. Tolman and L. H. Scharpen, unpublished data. (4) R. D. Gamer and R. V. Lindsey, Jr., J. Amer. Chem. SOC.,88, 2543 (1 966). (5) K. Hirota and Y . Hironaka, Bull. Chem. SOC.Jup., 37, 535 (1964) (6) J. E. Wollrab, “Rotational Spectra and Molecular Structure,” Academic Press, New York, N.Y., 1967, Chap. 4. (7) D. R. Lide, Jr., and D. Christensen, J . Chem. Phys., 35, 1374 (1961). (8) Y . Morino and E. Hirota, Nippon Kuguku Zusshi, 85, 535 (1964). (9) K. Hirota, Y . Hironaka, and E. Hirota, Tetrahedron Lett., 1964,1645. (10) Y. Hironaka, K. Hirota, and E. Hirota, ibid.,1966, 2437.

Table I. Line Frequencies (MHz) for Propene-D, and Propene-Do Species Frequency ( J = 0 .-t lol) CHaCHCHz 17439.45 CH3CDCHz 17139.02 s-CHZDCHCH~ 16833.OO U-CHzDCHCHz 16377.17 cis-CH3CHCHD 16769.80 ~FU~S-CH~CHCHD 16090.22

differs from theirs primarily in the use of a spectrometer designed for more routine operation, and computer control for most of the spectrometer adjustments, the data acquisition, and the error analysis. This greatly simplified the analytical procedure from an operator viewpoint and increased the accuracy of the measurements. We have also considered and determined the small contribution from power saturation effects and line width differences neglected in the earlier work. Additionally, we have determined from microwave measurements the amount of propene-do (CIH,) in each sample; with one exception, these data agree well with mass spectroscopy results. Finally, the error analysis given in the present paper is much more extensive. The main purpose of our error analysis was to determine the relationship between the accuracy limits calculated from the reproducibility of repeat measurements carried out over a period of time and the accuracy limits which could be expected on the basis of signal noise alone. Digital processing of signals with the computer was important since this permitted numerical evaluation of the noise statistics. EXPERIMENTAL

Apparatus. A Hewlett-Packard 8460A MRR spectrometer was used for the analysis. The computer used was a Hewlett-Packard 21 16B. Assembler language subroutines controlled the microwave frequency, the Stark modulation voltage, and analog-to-digital conversion of signals. Programs used to control the measurement sequence and to call the interface subroutines were written in BASIC. The major benefits accrued through use of the computer was the increase in signal measurement accuracy obtained from digital averaging of signals, and the ability to quantitatively estimate signal noise. It proved quite convenient, however, to use the computer to automate the measurement sequence as much as possible, to reduce the data to the desired final form, and to perform statistical calculations necessary for estimating error limits. A combination of electronic filtering and digital time averaging was used for processing signals. The spectrometer synchronous detector gain, adjustable in calibrated IO-dB steps, was always set to obtain a signal close to the I-volt full scale value. A detection system bandwidth in the range 1 to 10 Hz was set to keep the signal plus noise excursions below the 1-volt input maximum for the analog-to-digital converter linking the signal to the computer. After setting a microwave frequency, signal sampling was delayed for a period equal to 7 times the detection system response time to permit the signal to stabilize. Thereafter, the time between samples was twice the response time. The total time used for time averaging was varied from seconds to several minutes, depending on signal magnitude, in order to keep the relative precision of measured signals approximately the same for all measurements. Reagents. The partially deuterated propene samples were obtained from deuterium exchange in C H 3 0 D at 0 “C using homogeneous catalysts of Pt, Rh, and Ni ( 4 ) . After the

ANALYTICAL CHEMISTRY, VOL. 44, NO. 12, OCTOBER 1972

2011

~

~~

~

~~~~

Table II. Relative Factors Multiplying Experimental Signal Ratios to Obtain Concentration Ratios of Propene-D1 Subspecies to Propene-Do Factor CHsCDCHz s-CH~DCHCH~ u-CHDCHCH~ c~s-CH~CHCHD ~ ~ u ~ s - C H ~ C H C H D 0.997 0.994 0.988 0.992 0.985 Wb 1.033 1.073 1.133 1.082 1.173 V2 1.o 1.074 1.074 1.o 1.0 f” 0.999 1.008 0,997 0.991 1.004 u2 1.120 1.105 1.098 1.114 1.085 (ABC)”2 Power saturation 1.003 1.004 1.006 1.002 1.010 “Line width” 0.971 0.964 0.978 0.972 0.976 Total factor 1.122 1.235 1.295 1.155 1.241

(z)

Table 111. Isotopic Composition of Propene Samples from Mass Spectroscopy Analysis Sample DO D1 DZ Da D4 D 5 75 23 2.5 0.1 ’*(’) 64 30 5 0.4 0.1 Pt(2) 2.5 0.4 0.1 Rh(1) 55 32 11 Rh(2) 27 31 24 12 4.5 1 Ni 89 10 0.6 0.1

exchange, preparative gas chromatography yielded pure propene samples which were stored in 30 cm3 stainless steel cylinders. Five samples were made-two obtained with Pt catalysts, two with Rh, and one with Ni. A more detailed description of sample preparation will be reported elsewhere (3). Procedure. Microwave spectra generally are characterized by a number of narrow absorption “lines.” For a particular line, the output signal S from a conventional microwave spectrometer depends on the absorption coefficient y of the line S = GyL,

(1)

where L, is the effective absorption path length and G can be considered a proportionality constant which depends upon spectrometer operating conditions. In practice, G can be treated as a constant if the rectified dc current produced by the microwave detector is maintained at a constant value. The frequency dependence of y is Lorentzian in nature. A line is, therefore, characterized by the frequency at which peak absorption occurs (which is independent of mixture composition), the absorption coefficient at the peak yo, and the half-width of the line at half-maximum intensity Av. The effective length is related to physical length by Le = L(X,/X) where A, and X are, respectively, the radiation wavelength in the waveguide and in free space. The expression for yo is 70 =

N[8(?rh)8’2/3~(kT)6’2](ABC)1/2fo X

+ 1)pij2v2/Av (2)

e x d - WR/kT)(2J

where Nis the number of molecules per cm3, h is Planck’s constant, k is Boltzmann’s constant, c is the speed of light, T is the temperature in OK, W , and J are, respectively, the rotational energy and total angular momentum quantum number of the lower energy level of the transition, v is the absorption frequency, Av is the absorption line half-width at half-maximum intensity, and p i , is the dipole moment matrix element for the transition. A, B, and C are the molecular rotational constants, andf, is the fraction of molecules in the occupied vibrational state. We calculate that y o = 4 X lo-’ cm-l for the C3Heline in a pure sample. From the expressions give above, it should be apparent that the partial pressure of a compound in a mixture can be 2012

expressed as a simple function of the peak signal magnitude S, and the line half-width p = k3,Av(X/X,)/T3~2

(3) where K can be considered a proportionality constant. The value of K would obviously depend upon the specific line used but could be determined empirically for a specific line from measurements of S,, Av, T, and the pressure for a pure sample. The value of K will be somewhat temperature dependent through the Boltzmann factor for rotational energy and f,. It should be mentioned that Harrington (11, 12) has proposed an alternative method for quantitative intensity measurements requiring a different experimental approach which permits quantitative intensity data to be obtained at just the peak absorption frequency. The major constituent in each sample was undeuterated propene. It was convenient to use the absorption line from this species as an internal reference. The ratios of signals from each of the five monodeuterium subspecies to that from the normal propene in the sample were the basic experimental data. Since pure samples of the deuterated species were not available, the empirical calibration approach described in the last paragraph could not be used. However, the signal ratios could be easily converted to the desired partial pressure ratios by multiplying these by factors accounting for dif, v 2 which ferences in the quantities L,, (ABC)1/2,f,,p L i j 2and appear in Equations 1 and 2 and by factors determined experimentally for power saturation and internal rotation effects. Values for X/X, depend only on the frequency and the absorption cell dimensions. For standard X-band cell dimensions, X/X, = [l - (6557/v2)]I’*. For computing f,, we followed Morino and Hirota (8) and assumed that only the torsional vibrational mode contributes significantly so that f, = 1 - exp(-hhvto,,/kT) where vtors = 95.3 crn-’. The temperature was 298 f 2 OK. For the transitions used here, the dipole moment matrix element equals pa, the dipole moment component along the principal axis of least inertia. The factors for pa were calculated assuming that differences result only from the small rotation of the principal axis orientation which occurs with a change in isotopic composition. This neglects possible differences in the magnitude of the total dipole moment between isotopic species. The first experimental step was the determination of the peak absorption frequencies and the factors for internal rotation and power saturation effects. The rotational constants given by Lide (7) predicted the frequencies of the J = 0-lol lines for all species to within 60 kHz. The peak frequencies obtained from plots of signal intensity as a function of frequency are listed in Table I. Sample pressure of 100 mTorr (monitored with a thermocouple type gauge) and detector crystal current of 100 p A were chosen as convenient though arbitrary operating conditions and were used through(11) H. W. Harrington,J . Chem. Phys., 46,3698 (1967). (12) Zbid., 49, 3023 (1968).

ANALYTICAL CHEMISTRY, VOL. 44, NO. 12, OCTOBER 1972

Table IV. Observed Distribution

(x)of Propene-D, Subspecies

Sample identification Species MI) Pt(2) Rh(1) Rh(2) 25.4 (0.3) 57.8 (0.6) 30.7 (0.4) 58.0(0.7)a CHSCDCHz 40.4 (0.5) 26.6(0.3) 34.2 (0.4) 26.2 (0.5) CHzDCHCHzb 17.5(0.3) 17,2(0.3) 7.9 (0.2) 8.0(0.2) cis-CH3CHCHD 17.0(0.3) 7.7 (0.1) 17.6 (0.3) rruns-CH3CHCHD 8.0 (0.2) Estimated uncertainties (one standard deviation) are in parentheses. See text for discussion of error estimate. b Data here are the sum of values determined for the sym- and usym-CHzDCHCHz species.

Ni( 1) 16.8 (0.4) 53.1 (0.7) 15.3 (0.3) 14.8(0.3)

5

out the analysis. The absorption line half-widths needed for estimating internal rotation effects were determined by adjusting the frequency to the half-maximum intensity points on both sides of the absorption peak. Except for the H a species, the effect of methyl group internal rotation splits the absorption lines into unresolved doublets. The splitting varies with isotopic species and reduces the peak intensity from that expected if no splitting were present by the factor

+

F = [ ( R / ~ A V ) 11-l ~

(4)

where R is the splitting and Av is the half-width at halfmaximum intensity for each component of the doublet. The observable half-width Avobs of the line depends on both R and Av :

Since the H a species line is theoretically not split by internal rotation, Avobs = Av for this species. We assumed that Av was the same for all species and then calculated R from Equation 4 and F from Equation 3. The appropriate multiplying factor for an experimental signal ratio was then the ratio of F for the do species to that for the appropriate dl species. Hirota (13) gives calculated values for the splittings of the do and the Hz and H5 species lines. Our measurements indicate the actual splittings are somewhat less than these. For example, we found 112 + 10 kHz for the dosplitting compared to the 132 kHz calculated value. The correction factors of interest here, however, are relatively insensitive to the absolute values of R and Av as could be expected from the form of Equation 3. In fact, ratios of the observed line widths AvObs are within 1% of the factors obtained in the manner described. The actual values of the half-widths were about 310 kHz. Although it is safe to assume that the power saturation coefficient (14) is the same for each of the isotopic species, differences in the microwave power needed to produce the same detector crystal current at each frequency required that a correction factor for power saturation be determined. The lines were almost free of saturation effects and the correction factors were expected to be small, however. At each absorption frequency, the power required to produce the crystal current set during the analyses was measured. With the assumption that the power saturation coefficient was the same for each species, the correction factor could be determined from measurements on just the propene-do line. The incident power was set in turn to the values found for the other species while a microwave bridge was used to keep the crystal current constant. The fractional change in the absorption coefficient of the propene-do line was measured which gave directly the correction factor accounting for differences in power saturation. The sample pressure was set to the same value used during the analyses. (13) E. Hirota, J. Chem. Phys., 45, 1984 (1966). (14) C . H. Townes and A. L. Schawlow, “Microwave Spectroscopy,” McGraw-Hill,New York, N.Y., 1955, pp 371-3.

Table V. Percentages of Propene-Do and Propene-D, from Microwave and Mass Spectroscopy Data Propene-do Propene-dl__ Mass Mass Catalyst Microwavea spec Microwave spec 73.7(0.7) 75 22.6(0.3) 23 30 R(l) R(2) 63.2(0.7) 64 29.3 (0.4) Rh(1) 52.7(0.7) 55 31.9(0.5) 32 Rh(2) 21.3 (1.0) 27 31.6(1.5) 31 W.8(0.8) 89 10.4(0.1) 10 Ni a The quantity in parentheses is one standard deviation.

Table I1 lists values for the factors combining to yield the total multiplier needed to convert measured signal ratios to the desired partial pressure ratios. The only significant sources of error in these are the experimental “line width” or internal rotation factors and the assumption that isotopic substitution does not change the total dipole moment. The first of these contributes an uncertainty estimated as 1 of the listed values. Although the uncertainty associated with the dipole moment assumption should be small, it is difficult to assess without resorting to Stark effect measurements and was neglected in computation of the uncertainty estimates discussed below. After these preliminary measurements, the analysis reduced to measurements of the peak signal intensities for each species. Measurements were made in a standard spectrometer configuration. Esbitt and Wilson (15) give a detailed account of factors affecting the precision and accuracy of relative intensity measurements. We considered a number of these above and, wiih the 8460A, some do not apply or are accounted for in the design of the spectrometer. We chose a measurement sequence which would eliminate systematic errors from the remaining two factors which are variations in base-line signal voltage (or “pickup”) with microwave frequency and possible changes in sample composition and total pressure during the time of an experiment (due to absorption and desorption of gases at the walls of the sample chamber). Esbitt and Wilson (15) cited determination of the base-line signal voltage, particularly with weak signals, as a major source of uncertainty in microwave spectroscopy intensity measurements. Although we measured intensities of lines with absorption coefficients as small as 5 X 10-9 cm-’, determination of the base-line correction proved a minor inconvenience rather than a major problem. Variations in the base-line signal voltage over the time required to perform a sequence of measurements such as that described below was found to be insignificant. Therefore, the base-line voltage could be measured prior to introducing the sample and subtracted from the signal measured with sample present. To account for possible change in sample composition or pressure, the signal at the frequency of the d 1’me was (15) A. S. Esbitt and E. B. Wilson, Jr., Reo. Sci. Imfrum., 34, 901

(1963).

ANALYTICAL CHEMISTRY, VOL. 44, NO. 12, OCTOBER 1972

2013

Table VI. Experimental Ratio of asym- and sym-CH2DCHCH,Percentages (Theoretical Ratio is 2) Rh

Pt 1

2

1

2

1

Ratio

1.99 (0.06)

1.99 (0.05)

1.96(0.04)

2 . 0 0 (0.05)

1.95 (0.06)

measured just before and just after the signal from a dl species line. The average of these two values for the do Signal divided into the dl signal gave the desired ratio. The observed variations of signal magnitudes and total pressure was sufficiently small that this linear interpolation method removed any systematic error from this cause. The determination of signal ratios was straightforward. First, the base-line signal ioltages at the line frequencies were measured serially four times. The sample was introduced and the signal ratios were measured serially; again four repetitions were done. The standard deviations of the measured ratios in a single sequence such as this could not be used as a measure of the accuracy since the uncertainty in the baseline voltage enters as a systematic factor. To “randomize” this, the entire sequence beginning with the base-line determination was then repeated four times, using a fresh sample each time, and the data for the individual determinations were averaged. This procedure was used for each of the five samples. The ratio of the signal from the propene-doline in a partially deuterated sample to that from a sample of pure propene of normal isotopic composition gave directly the fractional amount of propene-do in a sample. The pressure was adjusted to the same value for each measurement. This number combined with the sum of the partial pressure ratios of propene-dl subspecies to the propene-do component determined the fractional amount of propene-dl in a sample.

RESULTS AND DISCUSSION Table I11 gives the overall isotopic composition of the five samples analyzed. Table IV gives the results of the microwave analysis for the amounts of the propene-dl, normalized so that the sum for each sample is 100%. Evaluation of the standard deviations given in parentheses is discussed below. Table V compares the microwave and mass spectrometer values for the amounts of propene-do and total amounts of propene-dl. The two techniques yield essentially identical results except for the amount of propene-do in the Rh(2) sample. The discrepancy is probably due to the fact that this sample contained a considerable amount of multiply substituted products (propene-d,, n > 1 ; see Table 111) which complicated the mass spectroscopy analysis. As discussed above, signal ratios were measured four times after each determination of the base-line signal. The four observations permitted calculation of an average value R, and its standard deviation u,. A value of u, estimates the random errors associated with the measurement with sample present but not those associated with the base-line measurement. Our procedure called for repeating this sequence four times. The average of four R, values then provided the best estimate of an intensity ratio. The standard deviation of this average, OAV (computed from the four observed R, values), provides an estimate of random errors including those associated with the base-line measurement, and thus it is a valid estimate of the nonsystematic errors in the experimental ratios. We should also be able to estimate UAV from the values found for u,. Random errors influencing the two measurements, the base-line determination and the signal measure2014

Ni

Catalyst

0

ment, should be uncorrelated and equal. It follows then that the variance of R, should be 2 ~ , 2when the base-line uncertaintyis included. estimate ofuAvis then (Test =

[~ZU,’]’’~/N

(5)

where in our case N = 4 and the sum is over a set of four values for u,. Our data provided 25 values for the ratio uAv/uest. The average of these was 1.3 with a standard deviation of 0.2. The range was 0.2 to 2. Since UAV and uest seemed to be essentially equivalent on the average, we chose to use their rms value URE = (UAV’ ~eat~)/2)”’ as a measure of the random errors. Standard deviations of the signals were calculated during the digital averaging periods. These were used to calculate estimates us for the standard deviations of the experimental intensity ratios, comparable to the quantities u, defined above. The average of the 100 values for the ratio u,/us was 1.7 with a standard deviation of 0.2. On the average, then, the scatter in the values for intensity ratios is somewhat larger than would be predicted strictly on the basis of the nominal uncertainty in the signal due to noise. This would indicate that other sources of error contribute slightly to the uncertainty of the ratio measurement. Our estimates for us may be too small since the signal sampling rate was too fast for the detection system response times employed. Separate experiments indicated that us would be increased by at most 15% if a slower sampling rate had been used. Part of the difference between ua and uscould thus be due to this factor. The correction factor for internal rotation splittings, discussed above, contributed the only significant and estimatable systematic error affecting the intensity ratio data. The standard deviation in Table IV estimating the total errors was USE*)^'^. calculated from utotsl = RE' Data for the H4 and Ha species provided an internal check of the analytical method and the error estimates. The theoretical ratio H4/H3is 2. The average value of the experimental ratios for the five samples, listed in Table VI, is 1.98. The standard deviation of this average is 0.02 when calculated from the experimental error estimates included in Table VI, compared to 0.01 when estimated directly from the five observed values. We restrict our discussion of the experimental deuterium distribution to a qualitative comparison with data reported previously by Hirota and coworkers (9, IO). Their exchange reactions were carried out using heterogeneous catalysts (Pt, Ni, and Pd) and DzO or D2as the source of deuterium as opposed to homogeneous catalysts and C H 3 0 D in the present work. When the experimental uncertainties are taken into account, the subspecies distribution in our sample obtained with a Ni catalyst was identical to that in their sample formed over a Pd catalyst with D 2 0 as the deuterium source. Similarly, our result with Pt catalyst was essentially identical to theirs with Ni, DzO. Our data for Rh catalyzed exchange do not match any of the subspecies distributions reported by them.

ANALYTICAL CHEMISTRY, VOL. 44, NO. 12, OCTOBER 1972

+

+

We expected and found that our data would show the effect of improvements in microwave spectroscopy instrumentation which have developed during the five-year period since the earlier work was reported. Because of differences in the amount of propene-dl in the samples analyzed and the temperature at which the measurements were made, the weakest line analyzed in the earlier work (10) (y 'v 3 X cm-l) was a factor of 6 stronger than that studied here (y = 5 X

cm-l). On the average, the standard deviations reported previously are a factor of 8 larger than we report here.

RECEIVED for review January 17, 1972. Accepted May 26, 1972. Contribution No. 1899 from the Central Research Department, E. I. du Pont de Nemours & Company, Experimental Station, Wilmington, Del.

Curcumin Method for Spectrophotometric Determination of Boron Extracted from Radiofrequency Ashed An imaI Tissues Using 2-Ethy I- 1,3-Hexa nedio1 James W. Mair, Jr., and Harry G . Day Department of Chemistry, Indiana University, Bloomington, Ind. 47401 Work on the nutritional significance of boron in animals required the development of a sensitive and accurate method for its determination at the submicrogram level. The method developed requires gentle combustion of animal tissues in a low temperature radiofrequency excited oxygen plasma followed by extraction from a 1N HCI solution of the ash using 2-ethyl-1'3hexanediol in chloroform (10% v/v). Boron in the organic phase i s converted to the highly absorbing rosocyanin complex using glacial acetic acid (0.375% w/v) followed by concentrated sulfuric acid. The concentrate i s diluted with 95% ethanol and spectrophotometrically read at 550 nm vs. a reagent blank. Beer's law is obeyed down to 0.002 pg/ml, and the method exhibits a total error of about 10% over concentrations ranging between 0.002 and 0.020 pg/ml. Data show that 100.3*5.1% of the standard Na2B407.10H 2 0 added to the unashed tissue samples was recovered. The use of XE-243 boron-specific resin proved to be a convenient, quantitative means for concentrating as little as 1pg of boron from large volumes of solution.

SINCETHE 1950's when Spicer and Strickland ( I ) demonstrated the utility of curcumin for boron determinations, many attempts have been made to improve the use of this reagent. In the original work, boron was separated from interferences by distillation as methyl borate and retained in platinum dishes with glycerol. After destroying the glycerol during the fusion, the highly absorbing rosocyanin complex was formed in the dishes by addition and drying of an acetone and water solution of curcumin. The complex was then extracted into o-chlorophenol and its absorbance read at 550 nm. This procedure was plagued with errors due largely to the numerous manipulations. Hayes and Metcalfe ( 2 ) modified the method by forming the rosocyanin complex in a mixture of 3 ml of glacial acetic and 3 ml of concentrated sulfuric acids. The strong acid protonated the curcumin to facilitate rosocyanin formation. Excess protonated curcumin unfortunately exhibited a spectral interference, but this could be removed by dilution of the concentrate with 95% ethanol in the Hayes and Metcalfe ( 2 ) method and by an ammonium acetate-acetic acid buffer

in the method by Grinstead and Snider (3). Uppstrom ( 4 ) used propionic anhydride to eliminate water, followed by direct analysis with the acid mixture. These methods all failed to achieve the degree of sensitivity possible with the rosocyanin complex because of the suboptimal reaction conditions which were employed. In the method to be described herein, direct standardization with primary standard borax is possible without prior chemical manipulation to simulate sample treatment. Standards need not be exposed to the sample ashing conditions because the radiofrequency method of sample ashing which was used enables boron to be completely recovered from the biological matrix. Complete recovery of boron permits the maximum sensitivity of the rosocyanin complex to be realized. EXPERIMENTAL

Apparatus. Polypropylene test tubes (Falcon Plastics No. 2059 size 17 x 100 mm) were used as vessels for tissue storage, for extraction, and for rosocyanin formation, and were effective in avoiding contamination. Combustion of animal tissues was accomplished using a Trapelo (formerly Tracerlab) low temperature biological sample asher Model 600. All spectrophotometric measurements were made using a Carl Zeiss PM QII spectrophotometer. Reagents. All standard boron solutions were prepared from reagent grade NazBIOi.10 H20. Solutions of 2-ethyl1,3-hexanediol (Aldrich) in chloroform (Allied Chemical) were 10% vjv. Curcumin from Eastman Organic Chemicals was recrystallized once from ethanol and used in glacial acetic acid (0.375 w/v). Procedure. Animal tissues were freeze dried and compacted into disks inch in diameter by inches thick using a pellet press and an applied pressure of approximately 4000 pounds. This step is optional, and serves only to allow large quantities of tissue to be handled more easily. Depending on the boron concentration anticipated in the sample, as much as 4 grams of the dried tissue disks were placed on aluminum foil squares or in silica dishes and inserted into each radiofrequency combustion chamber. These samples

(1) G. S. Spicer and J. D. H. Strickland, Anal. Chim. Acta, 18, 231 (1958).

( 2 ) M. R.Hayes and J. Metcalfe, Analyst (London),87,956 (1962).

R.Grinstead and Sigrid Snider, ibid., 92, 532 (1967). (4) Leif'R. Uppstrom, Anal. Chim.Acta, 43,475 (1968). (3) Robert

ANALYTICAL CHEMISTRY, VOL. 44, NO. 12, OCTOBER 1972

2015