of analysis and had a zero uncertainty. The K , factor was then calculated. The results of the analysis are given in Table IV. It can be seen that the results are very good. The uncertainties for the individual analyses are based on count rate. statistics. The uncertainties reported for the mean values are the standard deviations of the mean for each set. It is likely th'at the improvement in the data presented in Table IV, relative to bovine liver and orchard leaves, is due to the elimination of the flux inhomogeneity from the calculation. Also a small uncertainty in geometry is still present in the analysis of the bovine liver and orchard leaves which is obviously not present in the laboratory prepared unknowns. There are three primary advantages to using the Kn factor correction. Geometry and dead time corrections are simple for short lived nuclides for a nondestructive analysis. Accurate standard and sample placement during counting is not important (This means that a sample with a high dead time can be moved away from the detector without noting its location relative to the detector). The
method is fast since no special sample pretreatment is required. We presently use this method of Kn factor correction routinely in our laboratory. Several hundred samples have been analyzed in this manner. The method is rapid (generally 20-30 samples can be analyzed per day), and has been used on atmospheric and sea water samples.
ACKNOWLEDGMENT We are grateful to the nuclear reactor staff a t the Rhode Island Nuclear Science Center for providing space and facilities for these analyses. We also wish to acknowledge James L. Fasching of the University of Rhode Island, Kingston, R.I., for making a computer program, which analyzed the gamma ray spectra, available to us. Received for review May 29, 1973. Accepted October 26, 1973. This research was supported by the Office of the International Decade of Ocean Exploration, National Science Foundation, under NSF Grant GX33777.
Determination of Phenols by Fluorine-19 Nuclear Magnetic Resonance of Hexafluoroacetone Derivatives Floyd F.-L. HO Research Center, Hercules Incorporated, Wilmington, Del. 19899
Our previous study of the application of hexafluoroacetone to the characterization and quantitative measurement of alcohols has been extended to the determination of phenols. It was found that the F-19 resonance of the hexafluoroacetone-phenolic adducts can be measured with high sensitivity and resolution. All six isomers of dimethylphenol can be determined in a mixture at low concentrations. The dominant effect on the fluorine chemical shift of the adduct is steric interaction with the ortho substituents on the phenols; electronic and resonance effects from meta and para substituents provide secondary differentiation. Equilibrium constants of adduct formation were determined for several ortho alkyl phenols. Although reaction with most phenols is quantitative, caution should be exercised in the case of phenols with bulky ortho alkyl groups or strong electron-withdrawing substituents.
The determination of hydroxyl has been reviewed generally in two recent monographs ( I , 2 ) . In analyses for phenols, by far the most common procedures are based on titration of the phenolic hydroxyl with various chemical reagents. Recently, we have demonstrated that hexafluoroacetone reacts readily and quantitatively with common primary and secondary hydroxyls of both monomeric and polymeric materials (3).Measuring the fluorine NMR signal of these hexafluoroacetone adducts has the advantages of increased sensitivity (because of the replacement of a
single hydroxylic proton with 6 fluorines) and much greater resolution. This report describes the extension of this technique to the characterization and quantitative measurement of phenols.
EXPERIMENTAL The F-19 NMR spectra of the HFA adducts were obtained on a Bruker HFX-90 instrument, as described previously ( 3 ) .The operating frequency for fluorine resonance is 84.65 MHz. The ambient probe temperature was 25 "C. Hexafluoroacetone (HFA) was obtained from Pierce Chemicals, Rockford, Ill., in disposable cylinders (bp = -28 "C). Preparation of a stock reagent of HFA in ethyl acetate was described earlier ( 3 ) . Phenol and substituted phenols were reagent grade material purchased from either Eastman Organic or Aldrich Chemical Co. An NMR spectrum and a gas chromatogram were taken on each phenol to check its purity. Several o-alkyl phenols used in this study were further purified by fractional distillation at reduced pressure. The fluorine resonance of n-butyl trifluoroacetate, obtained from PCR, Inc., and purified by factional distillation, was used as an internal standard to provide a fluorine resonance signal for instrument integral calibration in quantitative measurements. The normal procedure for sample preparation involves weighing the phenol and internal standard (to 0.00005 gram) directly into a 1.00-ml volumetric flask, followed by dilution to the mark with HFA/EA reagent. Spectra taken immediately after mixing the sample solution showed that the reaction (Equation 1) is fast and quantitative with most phenols. OH
0 C F3 CCF,
+
R
I
CF, C-CF,
I
0
Academic Press, London, 1972. ( 2 ) "The Chemistry of t h e Hydroxyl Groups," S. Patai, Ed., lnterscience Publishers, New York, N.Y., 1971. (3) F. F.-L. Ho, Anal. Chem., 45, 603 (1973). (1) Stig Veibel, "The Determination of Hydroxyl Groups,"
496
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However, phenols with bulky substituents in t h e ortho position require longer reaction times. For example, t h e reaction with 2tert-butylphenol requires 24 hours a t room temperature t o reach equilibrium. Equilibrium constants for several o-alkylphenols were determined (Table III), using t h e general expression:
where CJ and c are molar concentrations of excess hexafluoroacetone ( a sharp singlet observed at 7500 Hz from H F B ) and phenolic adduct, respectively; b , is t h e molar concentration of phenol initially weighed into rhe flask.
RESULTS AND DISCUSSION Isomer Analyses. Several NMR procedures for characterizing isomeric phencils in a mixture have been described in the literature. Dietrich, Nash, and Keller ( 4 ) found that individual hydroxylic proton signals of phenols in a mixture dissolved in hexamethylphosphoramide (HMPA) can be resolved. Crutchfield, Irani, and Yoder ( 5 ) reported that characteristic patterns from the resonances of the protons o n the carbon atoms in the cr-position relative to the aromatic ring can yield information concerning the ortho:para ratio in alkylphenols. Lindeman and Nicksic (6) used acetate methyl resonances of the acetate derivative to determine ortho and para isomers. Konishi, Mori, and Taniguchi (7) differentiated isomers by observing the fluorine resonance of the corresponding trifluoroacetates. After comparing these methods with the one described in the present study, we believe that measurement of the fluorine resonance of the HFA derivative generally has the advantages of higher resolution, higher sensitivity, and easier correlation of chemical shift data with structural parameters. To illustrate the excellent resolution and sensitivity obtained with the HFA derivatives, a spectrum showing all six isomers of dimethylphenol is shown in Figure 1. This spectrum was run on a solution prepared by introducing 1- to 5-milligram quantities of each dimethylphenol isomer directly into a standard KMR tube containing about 1.0 ml of reagent. Each isomer in the mixture gives a sharp and characteristic singlet. Interestingly, these isomers can be roughly grouped into 3 classes based on substitution in the ortho position. Those having no ortho substituent appear a t a higher field; those having one ortho methyl appear in the center, and the one having two ortho groups is observed at a lower field. The substituents at the meta and para position provide a secondary differential. To demonstrate further the primary influence of ortho substituents on chemical shift, a limited series of phenols with different degrees of substitution was examined. The results, summarized in Figure 2? show that the dominant effect is indeed due to the substitution at the ortho position, with the magnitude of the shift increasing with the bulk of the substituent. In general, substitution a t the mela and para positions provides only a secondary influence (represented approximately by the length of the horizontal bar in Figure 2). For example, with respect t o unsubstituted phenol, the p-nitrophenol adduct appears a t only 0.05 ppin to lower field and the p-methoxyphenol a t only 0.01 ppm to higher field. These relatively small shifts suggest that the electronic and resonance properties of the substituents on the phenolic ring have little effect on the observed fluorine chemical shift of the HFA adduct. (41 M W Dietrich J . S . Nasti, and R. E. Kelier. Anal. Chem.. 38, 1479 (1966) ( 5 ) M . M . Crutchfieid, R . R . lrani, and J. T. Yoder, J. Amer. Oi/ Chem. Soc.. 41, 129 (1964) ( 6 ) L. P. Lindernan and S. W Nicksic. Anal. Chem.. 36, 2414 (1964). ( 7 ) K . Konishi Y Mort. and N . Taniguchi. Analyst (London), 94, 1002 (1969)
2.6
19F NMR spectrum of hexafluoroacetone adducts with all six dimethylphenol isomers (Field increases from left to
Figure 1. right)
PHEYOL
I
2-METHYL
I
2-ETHYL
I
2-ISOPROPII
I 2,6-DIMiTHOXYL
2,6-DIYETHYL
I
I
2 -A- B U T Y L
2-METHOXYL
I
I
I
I
86.00
I
1
85.00
8 PPM
,
#
t
84.00
,
l
83.00
FROM h F B
Figure 2. Summary of chemical shifts of phenols with various
substituents On the other hand, meta and para substituents can profoundly modify the reactivity of the phenolic hydroxyl group toward HFA. For instance, it was observed that the equilibrium constant of Equation 1 with p-nitrophenol is almost one order of magnitude smaller than that with phenol. A study of the effect of substitution at the meta and para positions on the reactivity of the hydroxyl group with HFA is in progress and will be reported elsewhere shortly. It is, nevertheless, the ortho substituents which dominate the chemical shift of the HFA gdduct. The reproducible and highly resolvable chemical shifts of the HFA adducts provide a sensitive tool in analyses of phenols. This ortho substituent effect evidently arises from steric interaction, although the detailed mechanism is not clear at present. A major contribution can perhaps be visualized as arising from the magnetic anisotropy of the aromatic ring current. The CF3 groups in a HFA adduct can assume various positions relative to the ring, by virtue of free rotation along both of the chemical bonds in the CO-CeH5 moiety. In ortho substituted alkylphenols, the A N A L Y T I C A L C H E M I S T R Y , V O L . 46, NO. 4, A P R I L 1974
497
Table I. Chemical Shifts of HFA and HMPA Derivatives of Phenols Compounds
'OF-HFAa
Phenol 3,5-Dimethyl 3,4-Dimethyl 2,5-Dimethyl 2,4-DimethyI 2,3-Dimethyl 2,6-Dimethyl
Table 111. Equilibrium Constants of HFA with o-Alkylphenols in EA at 25 "C
IH-HMPA~
83.855 83.820 83,852 84,197 84.245 84,339 85 ,036
10.30 10.02 9.93
2-Methyl 2-Ethyl 2-Isopropyl 2-tert-Butyl 2,6-Dimethyl
...
9 '97 10.02 9.20
a Parts per million downfield from hexafluorobenzene (HFB). Obtained from Reference 4.
MethvlDhenol Ortho" isomer Para isomer EthvlDhenol Ortio-isomer Para isomer
0'507[ A
0.319
=
0.464/ A
=
0.361
o-Alkylphenols
HFA~
TFAa
0.188
~
0.103 84'257[ 83 ,845
~
A =
A =
:
0.324
5.8
2.6
~
2-Methyl 2-Ethyl 2-Isopropyl 2-tert-Butyl 2,6-Dimethyl
~
~
log K
/
2.03 1.93 1.87 0.76 0.42
aorthoa
-0.13
...
-0.23 -0.52
...
pKab
10.15
10.28 ...
... 10.59
a Apparent uOrthoconstants
for substitution in phenols obtained from Reference IO. Dissociation constants from Reference 11.
0.412
'From Reference 7, converted from Hz (downfield from trifluoroacetic anhydride) to ppm, using the reported instrument frequency of 56.4 MHz. In ppm downfield from hexafluorobenzene. most favorable structure is that in which the CF3 groups occupy positions relatively far from the alkyl substituent, with the C - 0 bond in the C-O-C6& moiety almost in plane with the phenolic ring but on the side opposite from the alkyl group. This structure places the CF3 groups in the deshielding region of the ring current and correlates with a downfield shift. Because of steric interaction, this structure becomes more important with increasing size or number of ortho substituent. Thus, adducts with 2,6-dimethyl- and 2-tert-butylphenols are observed a t a progressively and substantially lower field. Notice in Figure 2 that the fluorine resonance of the omethoxyphenol adduct is found a t a slightly higher field than 2-methylphenol and that the corresponding signal from 2,6-dimethoxyphenol is even a t a much higher field. This again can be qualitatively accounted for by the ring current anisotropy. The hydroxyl in the adduct (see Equation l ) is more acidic and thus a good proton donor, favoring intramolecular hydrogen bonding with the electronrich oxygen atom of the o-methoxyl groups. A favorable position for the formation of this seven-membered, hydrogen-bonded structure is with the C-0 bond in the C-OC6H5 moiety making a large angle with the phenolic plane. This position places the CF3 groups above the plane and subject to the diamagnetic effect of the ring current, causing the adduct signal to be observed at a higher field. The chemical shift due to the difference in ring current anisotropy between these two extreme positions-Le., positions with the C-0 bond making the smallest and largest possible angles with the phenolic plane-is estimated to be about 4 ppm using the data of Johnson and Bovey (8). The chemical shift range in Figure 2 approaches this limit. Although the data obtained thus far appear to be consistent with an explanation based on a simple ring current model, other mechanisms which may contribute to the chemical shift in these compounds may be postulated, in(8) F. A . Bovey, "Nuclear Magnetic Resonance Spectroscopy," Academic Press, New York, N.Y., 1969, Figure 111-6, p 65.
498
107 84 73
Table IV. Comparison of Equilibrium Constants with Other Structural Parameters of o-Alkylphenols
Table 11. Comparison of Chemical Shifts of Triff uoroacetate and Hexafluoroacetone Adducts of Various Phenols Alkylphenols
Equilibrium constant (1.-mole- 1 )
o-Alkylphenols
A N A L Y T I C A L C H E M I S T R Y , V O L . 46, NO. 4 , A P R I L 1974
cluding the carbon-carbon bond interaction and/or short range polarization effects. A comparison of the chemical shifts of a mixture of dimethylphenols by proton NMR in hexamethylphosphoramide ( 4 ) and the present fluorine technique is shown i n , Table I. The resolution between a few pairs of isomers by the fluorine technique is slightly better. Of more importance in identifying the components of isomeric mixtures, however, is the pronounced ordering of the observed shift with respect to the structural parameters, in the phenol/ HFA adducts. A comparison between two fluorine NMR techniques, that using trifluoroacetate (7) and the present one using hexafluoroacetone adduct, is shown in Table 11. Although the data available for comparison are rather limited, higher resolution can clearly be obtained by using the HFA derivatives. This is perhaps due to the fact that the CF3 group in the trifluoroacetate derivative is further from the phenol ring, and thus less affected by the ring current or other localized effects. Equilibrium Constants. A brief study of the equilibria of Equation 1 for various phenols was undertaken to verify the applicability of the HFA technique to quantitative analyses of phenols. With water and most alcoholic hydroxyls, the equilibrium of Equation 1 normally lies far to the right (3, 9). For example, the enthalpy of hemiacetal formation of HFA with methanol in solution a t 25 "C was found to be -22.7 Kcal/mole (9). The present experiment offers a practical way to study the reaction of HFA with o-alkylphenols because the equilibrium under normal conditions lies in a convenient region for NMR measurement. Using the expression given in the Experimental section, a set of equilibrium constants for several selected o-alkylphenols was obtained, and the data are shown in Table 111. A gradual decrease in K is observed as the size of the substituents increases from methyl to isopropyl. However, the decrease in K is significantly greater when the substituent is tert-butyl or 2,6-dimethyl. With either of these two, steric interaction is severe. This dependence of K on (9) F. E. Rogers and R. J . Rapiejko, J . Amer. Chem. Soc.. 93, 4596 (1971). (10) G. B. Barlin and D. D. Perrin. Quart. R e v . , Chem. Soc.. 20, 75 (1966). (11) G . Kortum. W. Vogel, and K. Andrussow. Pure Appl. Chem.. 1. 187 (1961).
the size of the ortho-alkyl substituents parallels changes in other constants derived from the same structural differences, as may be seen from the data in Table IV. Again, only limited comparable data are available. As to the application of the HFA technique to quantitative analysis, it can be concluded that although it is applicable to most phenols, caution should be exercised in systems where there is a bulky ortho substituent or a strong electronegative group at the meta or para position. In such systems, the equilibrium does not lie too far to the right. This, however, can be partially overcome by the
presence of a great excess of free HFA. For example, to reach 99% reaction in a substituted phenol whose K with HFA is 100,a 1.0 molar excess of HFA should be present.
ACKNOWLEDGMENT The author thanks George A. Ward and Robert D. Mair for many helpful discussions, and Robert R. Kohler for experimental assistance. Received for review May 16, 1973. Accepted November 16, 1973. Hercules Research Center Contribution No. 1614.
Elemental Analysis of Whole Blood Using Proton-Induced X-Ray Emmission R. C. Bearse,' D. A. Close, J. J. Malanify, and C. J. Umbarger Los Alamos Scientific Laboratory of the University of California, Los Alamos, N.M. 87544
A technique for the analysis of trace elements in whole blood has been developed using proton-induced X-ray emission. Samples of 0.1 ml whole blood from humans and mice were dried, weighed, and then ashed in a plasma asher. Targets were prepared by placing drops made from the ash and a 400-ppm Pd solution onto Formvar backings. The samples were irradiated with 2.25-MeV protons, and the X-rays analyzed in a nondispersive X-ray detector. The elements Fe, Cu, Zn, Se, and Rb were detected with a precision of 7, 18, 7, 50, and 19%, respectively, in human whole blood. The precision of the technique was determined by statistical analyses of two different sets of 27 samples. The system was shown to be linear for variations in elemental concentration. The accuracy for determination of Zn was found to be within 1 0 % by comparison to atomic absorption spectrometry.
Although the study of trace elements in biological systems has been pursued for many years ( I ) , the increased awareness of trace metal toxicity has created a new interest in improved measuring techniques. The recent introduction of high-resolution nondispersive X-ray detectors has spurred a great deal of activity in X-ray fluorescence trace analysis. Several groups (2-4) have reported that proton-induced X-ray emission has great potential in trace analysis because many elements can be detected simultaneously and only small samples (-100 mg) are needed. Most of these papers, however, have been of a general and exploratory nature, and few deal with the details of a specific system for making measurements of a particular sample type. Most notably, previous workers seem to have relied on counting statistics as a measurement of precision, an approach that we have eschewed.
'
Visiting Staff Member from the University of Kansas, Lawrence, Kan. 66045 ( 1 ) E. J . Underwood, "Trace Elements in Human and Animal Nutrition." 3rd ed.. Academic Press, New York, N . Y . , 1971. ( 2 ) T. B. Johansson. R. Akselsson, and S. A . E. Johansson, NucI. Instrum. Methods, 84, 141 ( 1 9 7 0 ) . ( 3 ) F. C. Young, M . L. Roush, and P. G . Bergman, Int. J . AppI. Radiat. Isotop., 24. 153 (1973) ( 4 ) C. J . Umbarger, R. C. Bearse, D. A. Close, and J . J . Malanify, Advan. X - R a y A n a l . , 16, 102 ( 1 9 7 3 ) .
The purpose of this paper is to present details of a method of measuring trace elements in small volumes (0.1 ml) of whole blood using proton-induced X-ray emission. Blood was chosen because it is a physiologically important fluid that is easily sampled. We will discuss the basic principles of the technique, the blood sampling and preparation techniques, the apparatus used in analyzing the samples, the preparation of the proton beam, and the precision and accuracy of our final procedure. The principles of X-ray emission are simple. A heavyion beam, in passing through a thin sample, removes inner shell electrons with extremely high probability. The filling of these vacancies by outer shell electrons produces X-rays whose energies are characteristic of the element in which it is produced, and the number of X-rays is proportional to the number of atoms of that element present in the sample. Since the yield of X-rays per atom is a smoothly varying function of atomic number and bombarding energy (j), and since modern X-ray detectors are capable of resolving K a X-rays from the elements adjacent in the periodic table (for 2 > l l ) ,many elements can be measured simultaneously. Because of the multiplicity of X-ray lines ( e . g . KP) from each element and because of the possibility that some elements may be several orders of magnitude more prevalent than others, it may not always be possible to measure the presence of all elements without some prior chemical separation.
EXPERIMENTAL Sample Preparation. Capillary pipets. rinsed in heparin and air-dried, were used to draw 0.1-ml samples of whole blood from the sinus cavities of mice. H u m a n blood samples were drawn into 5-ml syringes, potassium oxalate was added, and the samples were repipetted with a n Oxford 0.1-ml autopipet. The samples were pipetted directly into I - m l borosilicate glass beakers t h a t had been previously weighed on a Torbal balance to a n accuracy of 0.2 mg. About 30 samples were processed a t one time. T h e filled beakers were arranged on a 7.6- by 15.2-cm borosilicate glass plate o n the base of a bell-jar system. a n d all beakers were repeatedly filled with liquid nitrogen until t h e blood was thoroughly frozen. With the beakers still containing liquid nitrogen, the bell jar was sealed and t h e system evacuated. The system was pumped for several hours, and a n ultimate vacuum of about 0.06 (5) R . C . Bearse, D. A . Close, J . J . Malanify, and C. J. Umbarger. Phys.
Rev.. 170, 1269 ( 1 9 7 3 ) .
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