Table 11. Unitary Chelate Effect for 1,lO-Phenanthroline and Bipyridine at 25 “C Reaction AGO (kcal/mole) A H o (kcal/mole) -.TASo (kcal/mole) CuPy2+ P = cuP2+ 2Py -4.2 -2.2 0.4 CuPy2+ 2P = CuP,a+ 4Py -8.9 4.9 -12.8 znPy,2+ P = znP2+ 2Py -0.9 -7.4 6.5 znPyaa+ 2P = znP2=+ 4Py -4.4 -5.7 1.3 CUPY,’+ B = CUB’+ 2Py -1.2 0.5 -1.7 CUPY,’+ 28 = CuBzz+ 4Py -4.3 4.7 -8.0 ZnPyZz+ B = ZnB2+ 2Py 1.6 3.5 -2.0 ZnPya*+ 2B = ZnB22f 4Py 0.0 10.9 -9.8
+ + + + + + + +
+ + + + + + + +
compared with a complex containing the corresponding unidentate ligand. It has been pointed out (45)that comparison of thermodynamic data to study structural effects is valid only for the mole fraction concentration scale. The thermodynamic quantities associated with the chelate effect for the interaction of Cu2+and Zn2+with P and P y are given in Table 11. Values for the chelate effect of bipyridine B, are also included where the log K and A H o values used for the interaction of Cu2+ and Zn2+ with B are those reported by Atkinson and Bauman (46)valid in lrn NaC104. The thermodynamic quantities given in Table I1 have been converted to the mole fraction scale by the method previously outlined (45). The results given in Table I1 indicate that in the case of bipyridine the chelate effect is entirely due to the favorable entropy changes, the A H o values being endothermic in all cases. For the reaction of Zn2+ with B the A H o values are (45) J. J. Christensen and R. M. Izatt in “Physical Methods in Advanced Inorganic Chemistry,” H. A. 0. Hill and P. Day, Eds., Interscience, New York, N. Y., 1968. (46) G. Atkinson and J. E. Bauman, Jr., Inorg. Chem., 1, 900 (1962).
endothermic enough that the chelate stabilization disappears when the thermodynamic quantities are put on the mole fraction basis. For the 1,IO-phenanthroline interactions the trend is largely reversed and the chelate effect is mainly due to favorable enthalpy changes. The stabilization of chelates due to favorable enthalpy changes has been previously noted for amine chelates (47). It is not presently clear why the bipyridine system apparently differs from the 1,IO-phenanthroline and other nitrogen containing chelate systems. For both ligands the chelate stabilization is significantly larger for the Cu2+complexes than for the corresponding Zn2+ complexes. ACKNOWLEDGMENT
The author thanks Donald C. Guthrie for assistance in performing the thermometric titrations. RECEIVED for review November 24, 1969. Accepted February 24, 1970. Appreciation is expressed to Shell Development Company for release of the material for publication. (47) A. E. Martell, Advan. Chem. Ser., 62,272(1967).
Improved Instrumentation for Phosphorimetry of Organic Molecules in Rigid Media Ruth Zweidingerl and J. D. Winefordner Department of Chemistry, Unirjersity of Florida, Gainesville, Fla. 32601 Phosphorimetry has previously been of limited use as a result of marginal precision and accuracy as well as difficulties and time of sampling. As a result of a rotating sample cell, a more stable source power supply, and a better solvent clean-up procedure, detection limits have been lowered more than one hundred-fold, precision and accuracy have been increased by more than ten-fold and a considerable reduction in time and effort in the sampling and measurement procedure has resulted as compared to phosphorimetric measurements with standard commercial equipment. The excellent precision and detection limits have been obtained not only in clear rigid solvents but also in cracked glasses and snowed matrices. The possibility of performing precise, accurate, sensitive, selective, rapid analysis in solventsformingopaque or densely cracked glasses greatly extends the usefulness of phosphorimetry-particularly to samples of biological interest.
PHOSPHORESCENCE IS A TYPE of photoluminescence in which radiation is emitted by an organic molecule following excitation of the molecule by ultraviolet or visible radiation of higher Research Triangle Institute, Research Triangle, North Carolina.
energy than the emitted radiation. It is experimentally differentiated from fluorescence, the other major type of photoluminescence, by its longer lifetime and lower energy. Phosphorescence is a true molecular property and so is useful in the identification of organic molecules. The mechanism of the production of phosphorescence is well-known ( I , 2) and will not be discussed here. Phosphorimetry is the analytical method involving the use of phosphorescence for quantitative analysis. Zander (3) and Winefordner, McCarthy and St. John (4) have described the use of phosphorimetry in analysis. Phosphorimetry has been used during the past decade for a limited number of analyses, e.g., the analysis of impurities in ~
~
~
~~~
~
(1) D.M. Hercules, “Fluorescence and Phosphorescence Analysis,” D. M. Hercules, Ed., Interscience, New York, 1966. (2) C. A. Parker, “Photoluminescence of Solutions,” Elsevier, New York, 1968. (3) M. Zander, “The Application of Phosphorescence to the Analysis of Organic Compounds,” Academic Press, New York, 1968. (4) J. D. Winefordner, W. J. McCarthy, and P. A. St. John, “Methods of Biochemical Analysis,” D. Glick, Ed., Vol. 15, Interscience, New York, 1967. ANALYTICAL CHEMISTRY, VOL. 42, NO. 6, MAY 1970
639
polycyclic aromatic hydrocarbons (5-9,in coal tar fractions (a), in air (9-11), and in petroleum fractions (12-14), the the analysis analyses of inhibitors in petroleum products (U), of pesticides, fungicides, oils, etc., in foods (16-20), and the analysis of amino acids and pharmaceuticals in biological fluids (21-29). It is clear that up to now phosphorimetry has been used only sparingly as an analytical tool and has been confined primarily to those cases where other analytical methods, such as fluorimetry and absorptiometry, give either no results or ambiguous results. In fact, fluorimetry and absorptiometry have generally been used for quantitative analysis which could be more selectively and sensitively performed using phosphorimetry. This is simply a result of the greater complexity and time required to perform an analysis and the poorer precision and accuracy of analysis (1 % for fluorimetry and absorptiometry us. 10 for phosphorimetry). These factors are discussed in more detail below. Phosphorescence is generally not observed in liquid solutions at room temperature because radiationless deactivation due to collisions which occur within the lifetime of the excited molecule result in low phosphorescence quantum yields (30) producing phosphorescence signals buried within the phosphorescence background noise (31). Therefore all analytical phosphorimetry studies are carried out in rigid media (3, 4). The criteria previously used to select a solvent for phosphorimetry have been: solubility of the analyte; low phosphorescence background (easy to purify); and formation of a clear rigid glass at 77 OK (boiling point of liquid nitrogen; a safe, inexpensive, optically transparent coolant). ( 5 ) M. Zander, Angew. Chem. Intern. Ed. Eng(., 4, 930 (1965). (6) M. Zander, Z . Anal. Chem., 226, 251 (1967). (7) E. Clar and M. Zander, Chem. Ber., 89, 749 (1956). (8) M. Zander, Erdoel Kohle, 19,279 (1966). (9) E. Sawicki, Chemist-Analyst,53, 88 (1964). (10) E. Sawicki, and H. Johnson, Microchem. J . , 8, 85 (1964). (11) E. Sawicki, T. W. Stanley, J. D. Pfaff, and W. C. Elbert, Anal. Chim. Acta, 31, 359 (1964). (12) N. K. Sidorov and G. M. Rodomakina, Uch. Zap. Saratousk Gos. Uniu., 69, 161 (1960). (13) H. V. Drushel and A. L. Sommers, ANAL. CHEM.,38, 101 (1966). (14) Zbid., 38, 19 (1966). (15) Ibid., 36, 836 (1964). (16) W. J. McCarthy and J. D. Winefordner, J. Assoc. Ofic. Agr. Chemists, 48, 915 (1965). (17) J. D. Winefordner and H. A. Moye, Anal. Chim. Acfa, 32, 278 (1965). (18) H. A. Moye and J. D. Winefordner, J . Agr. Food Chem., 13, ' 516 (1965). (19'1 Zbid.. 13. 533 (1965). 1 : IP
(15)
= 4PlP
Therefore, all analytical curves (log I p us. log C )have a slope of unity at low concentrations and a slope of zero at high concentrations of analyte. For an optically inhomogeneous medium, e.g., a snow or densely cracked glass where s > 0, the general expression is given by Equation 12. For low concentrations of analyte where k b 1 , Equation 12 reduces to:
lytes, s is independent of the analyte (do), Le., little change in particle size occurs with concentration. The illumination has been assumed diffuse and scatter within the sample diffuse. In practice, a directed beam is generally used for illumination and the second assumption is relied upon to meet the criteria of diffuse reflectance. The validity of this assumption depends on the extent of the scattering within the sample and the lack of any orientation of the particles. The use of non-diffuse illumination may increase the importance of the specular component of the reflectance. The sample was assumed planar with infinite lateral extension. This assumption is not valid for the small bore sample cells used in these studies. However, because of the good agreement between experimental and predicted analytical curves, no correction for edge effects was made (such a correction is constant only at low analyte concentrations and is a complex function of cell geometry and analyte concentration at high concentrations). The values of s may range from less than one to several hundred (52). The value of s can be determined by reflectance measurements from :
where R , is the reflectance of a sample of sufficient thickness that no further increase in thickness will change the reflectance, Le., Kb a . The value of k must be found independently in order to evaluate s. Kortum et al. (43) have experimentally verified Equation 20 using a didymium glass filter which they ground to varying particle sizes. The equations derived here describe the behavior of phosphorescence of organic molecules in a diffusely scattering (and clear) medium. The model assumed results in Equation 12 which describes the behavior of phosphorescence in solvents which form snows as well as clear rigid glasses and clear liquid solutions. No attempt has been made to account for absorption of the phosphoresence within the sample because it is usually extremely small.
-
EXPERIMENTAL Instrumentation. An Aminco-Bowman spectrophotofluo-
It k >> s, then I reduces to Equation 1 5 . If s >> k , then:
and so analytical curves (log I p us. log C) in inhomogeneous diffusely reflecting media should have a slope of unity at low concentrations, a slope of 0.5 at intermediate concentrations, and a slope of zero at high analyte concentrations. Validity of Assumptions. The illumination of the sample was assumed to be monochromatic to avoid complications from the wavelength dependence of the absorption and scatter. The scattering coefficient has been shown by Kortum and Oelkrug (51) to vary with “particle size” and “wavelength.’’ If s a l / A y ,then for (dz>”* < A, y = 2.6 to 3.6; for (&)1/2 = A, y = 1 ; and for ( d * ) l ’ Z > A, y = 0 to 1, where (dz)l’ is the root-mean-square particle diameter. This indicates that, for very small particles, a dependence characteristic of Rayleigh scattering is approached. Measurements of s made at different wavelengths should be compared with caution. For analytical concentrations of most ana(51) G . Kortum and D. Oekrug, 2.Nuturforsch., 19a, 28 (1964). 642
ANALYTICAL CHEMISTRY, VOL. 42, NO. 6, MAY 1970
rometer with an Aminco-Keirs phosphoroscope attachment, a 150-watt xenon arc lamp, and a potted RCA 1P28 multiplier phototube (American Instrument Co., Inc., Silver Springs, Maryland) was used. Unless specified otherwise, all measurements were made using the Aminco elliptical source condensing system (American Instrument Company). Polarization measurements were made using Glan Thompson polarizers. Phototube signals were measured with a low-noise-manoammeter described by O’Haver and Winefordner (53) and recorded with a Model A601R milliamp recorder (Esterline Angus, Indianapolis, Indiana). Spectra were recorded with an X-Yrecorder. A modification of the rotating sample cell described by Hollifield and Winefordner (44) consisting of a Varian A60-A High Resolution Nuclear Magnetic Resonance Spectrometer Spinner Assembly (Varian Associates, Palo Alto, California) mounted on a sample compartment light cover was used (see Figure 2). The pressure cap of the spinner assembly was covered with black tape to ensure a completely light tight sample compartment. The rotating sample cell was driven by nitrogen pressure from the liquid nitrogen storage Dewar, The pressure was maintained by passing a current through a nichrome coil inside the liquid nitrogen Dewar container to vaporize some of the liquid nitrogen. (52) H. C. Hamaker, Philips Res. Rep., 2, 55 (1947). (53) T. C. O’Haver and J. D. Winefordner, J . Chem. Educ., 46,241 (1969).
n --A
-C
Figure 3. Starter circuit for xenon arc lamp
u
Ci, Cz, C3-0.5 c 4
--D
IYr Figure 2. Schematic diagram of rotating sample cell assembly
A. Quartz sample cell B. Varian (909614-04) spinner assembly for NMR C . Aminco light cover mount D. Aminco quartz Dewar flask
The volume above the liquid nitrogen was used as a ballast. The nitrogen flow was regulated with a needle valve and monitored with a rotameter, The speed of sample cell rotation was measured with a Strobotac Type 1531 (General Radio Company, Concord, Massachusetts). The xenon arc lamp was powered by a Harrison Lab Model 6268A power supply (Hewlett-Packard, Palo Alto, California) operating in the constant current mode at 7.5 amps. The lamp was started using a circuit schematically represented in Figure 3 (many other circuits were constructed, but only the one in Figure 3 operated properly). Intensity fluctuations were less than 0.1 %, the limit of the measuring system. This stability is similar to the arrangement described by Green et al. (54). Where noted, an Aminco power supply furnished with the spectrophotofluorometer was used. Reagents. Reagents which were used as received were: DL-tryptophan, oxythiamine hydrochloride, sulfanilamide and 3-acetylpyridine (Nutritional Biochemical Corp., Cleveland, Ohio) ; toluene (Malhckrodt Chemical Works, St. Louis, Missouri); 2,2,4-trimethylpentane (isooctane), spectroquality (Matheson, Coleman and Bell, East Rutherford, New Jersey); propylene glycol, fluorimetric grade (Hartman, Leddon and Co., Philadelphia, Pennsylvania). Ethanol was purified by distillation through a five-foot column filled with helical coils and vacuum-jacketed and silvered. The ethanol was collected at a reflux ratio of 20 to 1. Water used as a solvent was freshly distilled. Procedure. Stock solutions of compounds were prepared in concentrations ranging from 10-lM to 10-4M depending upon their solubilities. [f the phosphorescence measurements could not be performed immediately, the stock solutions were stored at 20 “C. Analytical curves were prepared from successive dilutions of the stock solutions. A 1, 3, 1, 3, 5 , 3, slit arrangement with the elliptical condensing system was (54) M. Green, R. H. Breeze, and K. Bacon, Rev. Sei. Instr., 39, 411 (1967).
c 5 c 0
R1
Rz R3
SI SZ L1 Lz D TI Tz
pF (300 VDC) -1300 p F (300 VDC) -0.01 p F (30,000 VDC) -100 p F (300 VDC) -1 R -1KQ -1.8 K R -Momentary contact switch -15 ampere switch-to be closed only after lamp ignites -300 mH (10 A, hand wound on ferrite core) -30 mH (15 A, hand wound on ferrite core) -Diode (15 A, 300 PIV) -Autotransformer -10 : 1 Ratio transformer
used for all studies. All determinations were made in triplicate except near the limit of detection where 5 to 10 replicate samples were measured. The excitation and emission spectra were recorded with the identical slit arrangement. Spectra were not corrected (3, 4) for instrumental response. The limits of detection were determined from the signal-tobackground fluctuations by the statistical method of St. John, McCarthy, and Winefordner (55). A 1-second time constant was used in the manoammeter. All precautions of cleanliness were observed as described by Zweidinger, Sanders, and Winefordner (56). The measurements obtained for the propylene glycol and water (9:1, V/V) solvent system were performed with the Aminco lamp power supply. A stock solution of 1 toluene in ethanol was measured between unknown samples to compensate for source fluctuations. A high degree of cracking was induced in the propylene glycol-water mixture by the introduction of a small plug of quartz wool just above the optical path. Polarization measurements were made using polarized excitation. Correction for instrumental polarization was made by the method suggested by Azumi and McGlynn (57). If RII‘ and RL‘ are the vertical and horizontal components of the luminescence with horizontally polarized excitation and Iill and R L are the vertical and horizontal components with vertically polarized excitation, then :
The precision of the phosphorimetric measurements was determined by repeated sampling of the same solution. Measurements were made on solutions which formed clear glasses, snows and cracked glasses. Sample tube diameters were determined by introducing a known volume of liquid into the tube and determining its height in the tube (tubes had a constant bore). (55) P.A. St. John, W. J. McCarthy, and J. D. Winefordner, ANAL. CHEM., 39, 1495 (1965). (56) R. Zweidinger, L. B. Sanders, and J. D. Winefordner, Anal. Chim.Acta, in press. (57) T. Azurni and S.P. McGlynn, J. Chem. Phys., 37, 2413 (1962). ANALYTICAL CHEMISTRY, VOL. 42, NO. 6, MAY 1970
643
TOLUENE
CONCENTRATION,
moles/liier
Figure 4. Experimental analytical curves for toluene in ethado1 (A); propylene glycol-water (4:l; V:V) (B); and isooctane (C); at 77°K relative phosphorescence signal us. toluene concentration
The phosphoroscope can was adjusted to the greatest speed of rotation (about 15,000 rpm). The speed of sample tube rotation was calibrated with a strobe light against the readings of the rotameter (gas flow meter). Speeds from 450-rpm to 1400-rpm were obtained for the sample cell rotator. At the rather high speeds of the phosphoroscope can and the low speeds of the sample rotator, no difficulty was encountered with fluctuations in the readout due to beat frequencies. Particle size was estimated by counting particles per grid with a magnifier at 150X magnification.
RESULTS AND DISCUSSION Comparison of Experimental Results with Equation 12. Experimental phosphorimetric analytical curves for toluene in three differential solvents, ethanol, propylene glycol, and water, and isooctane, are shown in Figure 4. At 77 OK, ethanol is a clear rigid glass and scattering is minimal. Both isooctane and the propylene glycol-water mixture form snows. Isooctane snows are fine grained with 2-mm thick samples being visually opaque. The propylene glycolwater snows are densely cracked with particles large enough to be distinct to the eye and 2-mm thick samples are visually translucent. The particle sizes for propylene glycol-water were between 400 and 150 I.( and those for isooctane were less than 40 I.( which was the limit of the magnifier used. The latter was limited by the long focal length necessary due to the Dewar walls. The appearance of the isooctane snows changed at high concentrations of toluene to a more translucent snow, and at 0.1M many samples did not form snows but were only lightly cracked. It should be noted that the snows resulted in greater phosphorescence signals at low concentrations (the linear portion of the curves), whereas the rigid ethanol solvent resulted in higher phosphorescence signals at high concentrations. This agrees with limiting cases of Equation 12 (see Equations 14, 17, and 18). Several theoretically calculated analytical curves for toluene using Equation 12 are given in Figure 5. The variable parameters in Equation 12 were taken as: b E 0.2 cm (the bore diameter of the sample tube); t was estimated to be 200 at 265 nm by using room temperature measurements; and C was known from the experimental analytical curves. It was not possible to measure s with the instrumentation available. An estimated s of about 50 cm-I for propylene glycol-water 644
ANALYTICAL CHEMISTRY, VOL. 42, NO. 6, MAY 1970
TOLUENE CONCENTRATION,
moles/liter
Figure 5. Theoretical analytical curves calculated using Equation 12 for toluene: A. s = 0; B. s = 50; C. s = 100; D. s = 300; E. s = 500; andF. s = 800
and greater than 100 cm-l for isooctane solvents were obtained using the approximation method of Hamaker (52). By superimposition of the experimental and theoretical analytical curves for s = 0 cm-’, s = 50 cm-I, and s = 800 cm- for ethanol, propylene glycol-water, and isooctane, respectively, good agreement is obtained. The agreement is especially good for ethanol and propylene glycol-water mixtures. The experimental analytical curve of isooctane appears to be approaching the linear portion of the propylene glycol-water mixture at low concentrations. At high concentrations, the curve for isooctane appears to have a slope of about 0.5 as predicted. Small deviations between the theoretical and experimental analytical curves could result due to errors in measuring e and b. Of course deviations could result due to invalidity of the assumptions and ultimately the model. However, the theory predicts quite accurately the general shape of the curves. Also there is a good agreement of the low concentration portion of the curves (especially for ethanol and propylene glycol-water) and the high concentration region over which bending occurs. Such agreement is consistent with the model chosen. It is interesting to note that for any given concentration on the linear (low concentration) portion of the analytical curves, the snows give about a two-fold larger signal (actually the ratio of phosphorescence signal in cracked or snowed samples of sulfanilamide and tryptophan were 1.7 f 0.2). Also the analytical curves for snows deviate sooner from linearity than the glasses. However of great analytical consequence is the fact that “snows are essentially as analytically useful as clear rigid glasses.” The influence of sample cell diameter upon the phosphorescence signal is accurately described by Equation 12. However, it should be kept in mind that an increase in cell diameter also results in an increase in illuminated and measured sample area as well as an increase in path length, b, i.e., two dimensions change (the height remains constant). Polarization measurements of the snowed and clear rigid matrices were also made to verify the assumption of diffusely reflecting media. The phosphorescence polarization of 4.7 X 10-3M toluene in ethanol clear glass was -0.33 =k 0.04 and in 2.4 X 10-5Msulfanilamide in ethanol clear glass was -0.27 =k 0.02 which indicated essentially complete polarization in rigid glasses. On the other hand, the phosphorescence polariza-
E
w 80-
a
90 I
I
I
I
Figure 7. Experimental analytical curves : for sulfanilamide in isooctane: ethanol; 4:l - V:V (A); for 3-acetylpyridine in isooctane (B); and for oxythiamine hydrochloride in iso0ctane:ethanol; 4:l V:V(C)
-
SCATTERING COEFFICIENT,S, cm-’ Figure 6. Theoretical dependence of phosphorescence signal on degree of scattering for various absorption coefficients (k). Change of phosphorescence signal compared to signal from same analyte in diffusely illuminated clear rigid glass (s = 0)
tions of toluene and sulfanilamide in isooctane were essentially zero (-0.03 + 0.04 and 0.00 i 0.02, respectively) indicating complete depolarization and complete randomization of the particles in snows and cracked glasses. For phosphors with lifetimes less than 100 milliseconds, there may be a dependence of phosphorescence intensity upon speed of sample cell rotation. Because phosphorescence , t growth depends upon the growth factor 1-exp (- t / ~ )where is the exposure time and Tis the lifetime, the growth factor will be less than unity if Tis of the order of 7. It should be stressed however that the influence of speed of sample cell rotation on signal is quite complex because the measured phosphorimetric signal depends upon the time for growth, the phosphoroscope rotation speed delay time between excitation and measurement, the decay during measurement; these factors depend upon the size of sample cell, the geometry of the excitation and emission optics, and the direction of rotation of the sample cell (the sample cell rotates clockwise-away from emission monochromator entrance slit and so the time period for decay is about three times longer than the time for excitation). The results of this study can be summarized: the sample cell should rotate counterclockwise to obtain the greatest signal; prior to a measurement, the sample cell rotation should be varied to determine the variation of phosphorescence signal with speed; and assuming a speed dependence, the sample cell rotation speed should be adjusted to give the smallest variation of signal with speed: the nitrogen flow driving the spinner should be well regulated. The deviation of the phosphorescence intensity with changes in s (extent of scattering) is important to the analyst because s may deviate from sample to sample. In Figure 6, the theoretically predicted change in signal with respect to a diffusely illuminated clear rigid glass (s = 0) is shown to be very small at low values of k but large at higher values of k . This may explain the higher standard deviations observed for high concentrations of toluene in isooctane where the appearance of the matrix was variable. No difference in standard deviation is
observed for sulfanilamide and the appearance of the samples was uniform throughout the range of concentrations. Thus, if the molecule being analyzed does not change the nature of the snows by its presence in high concentrations, the value of s does not change sufficiently from sample to sample to influence the precision significantly. The results obtained for phosphorescence polarization indicate that snowed samples approximate diffusely reflecting media. The nearness of the approximation very likely depends on density of cracking or particle size consistent with the higher standard deviation for cracked glasses compared to snows. Cracked glasses did not show a more significant improvement in standard deviation when rotated than did the other types of media. This indicates that rotating cracked glasses does not increase their diffuse reflectance properties. This should not obviate the use of rotating sample cells for determinations in cracked glasses because the inner filter effect is reduced, sampling is simpler and more rapid, and the precision under these conditions is better than is generally expected for phosphorimetry of clear glasses using the commercial stationary sample cell holders (44). Beyond a minimum level of cracking, the behavior becomes predictable on the basis of diffusely reflecting media and the precision similar to that obtained for clear glasses. Analytical Studies. Analytical curves for toluene in ethanol, propylene glycol-water, and isooctane are shown in Figure 4. Analytical curves for sulfanilamide, 3-acetylpyridine, and oxythiamine hydrochloride in isooctane are shown in Figure 7. These curves are similar to those of toluene in isooctane. The range of linearity of these curves for snowed matrices should be noted (also see Table I). The instrumental system used in our studies had several improvements over instrumental systems previously used in phosphorescence studies. These improvements were : the rotating sample cell (Varian NMR spinner) resulted in greater precision and accuracy of measurement, was simpler and more rapid sample changing, and had a reduced inner filter effect; the Aminco elliptical condensing system and the constant current power supply (Harrison)reduced long term drift by nearly a 100-fold and short term fluctuations by about 10-fold; a highly stable, sensitive current detection system; and an effective clean-up and sampling procddure minimized contamination problems. As a result of the above improveANALYTICAL CHEMISTRY, VOL. 42, NO. 6, MAY 1970
645
~~~~
Table I.
~
Phosphorescence Characteristics of Several Organic Studied Molecules
Concentration range ( M ) of
Compound Toluene Toluene
near linearity 103
Slope of linear portion
Relative standard deviation"
Solvent Ethanolb 0.94 1.5 Propylene glycol: c water (9 :1, VjV) 103 0.76 2.5d Toluene IsooctaneC 103 0.89 0.96(6.1)' Sulfanilamide Isooctane:c Ethanol (4: 1, VjV) 105 0.96 0.86(1.1)a Oxythiamine Isooctane:" 102 1.01 1.9 Hydrochloride Ethanol (4:1, VjV) 105 1.03 1.9 3-Acetylpyridine Isooctanec For samples giving signals 5 times the limit of detection. b Formed clear glasses. c Formed snowed samples. d Harrison power supply not used. e Linear portion of the analytical curve. f Nonlinear portion of the analytical curye. 0 Limits of detection in this study. h Best previous limits of detection using standard commercial phosphorimetric equipment. * From Reference 58.
Linear correlation coefficient 0.9999
Limit of detection Pgimlg 0.03
Limit of detection pgimlh
...
...
0.9999 0.9998
0.03 0.02
0,9998
0.0004
0.012i
1.0000 1.oOOo
0.34 0.012
... 3.6i
...
Q
Table 11. Precision of Phosphorescence Measurements" for Clear Glasses and Snows Using Varian Spinner Assembly
Number of determinations
Relative standard deviation, Stationary Nature of random Stationary matrix orientation aligned Rotating 1.3 0.8 4 Clearb 8.7 6.0 0.5 0.8 6 Clearb 13.7 3.4 1.4 5 Crackedb 0.9 0.3 11 Clearb 3.1 2.8 1.0 10 Clearb 2.9 1.6 0.9 5 Snowc 3.6 0.8 0.6 10 Snowc 2.8 2.4 0.7 10 Snowc 2.7 a Phosphorescence measurements made on 1.6 X 1W6M SUIfanilamide solutions which gives a signal 5 orders of magnitude above phototube dark current. b Ethanol solvent. c Isooctane:ethanol, 4: 1, VjV mixture as solvent.
ments, much smaller phosphorescence signals could be measured. For example, the limit of detection was taken to be that concentration resulting in a signal two times greater than the background noise (this signal was about 10% of the phosphorescence background). In the past (4), this definition could not be used because of the large, variable phosphorescence background. I n Table I, limits of detection obtained for toluene, sulfanilamide, oxythiamine hydrochloride, and 3acetylpyridine in several solvents (ethanol-clear glass, propylene glycol-water-finely cracked glass and isooctane-snow) are given. These limits of detection are of the order of 100-fold better than the best previous limits of detection obtained with standard commercial phosphorimetric instrumentation, (see (58) L. B. Sanders, J. J. Cetorelli, and J. D. Winefordner, Talanta, 16,407 (1969).
646
0
ANALYTICAL CHEMISTRY, VOL. 42, NO. 6, MAY 1970
Table I). It should also be stressed that the present limits of detection were conservatively estimated, whereas many of the previous limits of detection were only possible under very limited conditions. The improved instrumental system used in these studies resulted in about a IO-fold increase in precision for clear rigid glasses as compared to the previously used standard commercial phosphorimetric system. The improved precision is primarily a result of smaller sample positioning errors and a more stable source. It should be stressed that the new rotating sample cell holder even when used in the stationary aligned mode (no rotation) gives nearly a ten-fold increase in precision. when the sample cell is rotated, a further improvement of about two-fold occurs in the precison (see Table 11). It is interesting to note that the precision for snowed and cracked samples is not significantly different from the ethanol clear, rigid glasses. The stationary randomly oriented sample cell measurements were considerably more precise than obtained by Hollifield and Winefordner (44) because of the close tolerances of the present rotating sample cell system. The greatest single advantage for using the rotating sample cell is the simplicity of positioning the cell. After the sample cell is cleaned and filled (requires about five minutes), the cell with the Teflon (Du Pont) turbine is simply dropped into placed and spun by flow of nitrogen gas. As a result of spinning the cell, the inner filter effect is also minimized. The major limitations to the use of phosphorimetry now seem to have been minimized. The improved detection limits and precision, the simplicity and speed of sampling, and the possible use of solvents forming inhomogeneous matrices should result in the use of phosphorimetry in routine analyses in biology and medicine.
RECEIVED for review November 10,1969. Accepted February 25,1970. Research was carried out as a part of a study on the phosphorimetric analysis of drugs in blood and urine, supported by a 1J.S. Public Health Service Grant (GM-11373-06).
