Capillary Cell for Phosphorimetry of Organic Molecules in Aqueous Solvents at 77°K R. J. Lukasiewicz, P. A. Rozynes, L. B. Sanders, and J. D. Winefordner’ Department of Chemistry, University of Florida, Gainesville, Fla. 32601 Analytical phosphorimetric studies of polar organic molecules in predominately aqueous solutions at 77 O K has been achieved by use of an open quartz capillary tube for the sample cell. Limits of detection (between 10-lOand 10-11gram) and analytical curves (linear over a 104-fold concentration range) for p-nitrophenol, sulfamethazine, and 2-thiouracil were measured and found to compare favorably with the best previously reported values using nonaqueous solvents and larger sample cells. The measurement of phosphorescence of molecules in biological systems (sample size required isonly about20pl)isnowpossible becauseof the use of a 90% water-10% methanol solvent which has a very small phosphorescence background. The larger long-lived luminescence background of the quartz capillary cells was minimized by the use of an excitation polarizer oriented perpendicularly to the sample cell length.
PHOSPHORIMETRY was introduced as a means of chemical analysis by Keirs, Britt, and Wentworth (I) in 1957. Since then, a number of publications have appeared describing both improvements in the technology of phosphorimetry and its applicability to chemical analysis. Several reviews (2-5) describing the use of phosphorimetry have appeared recently and contain extensive bibliographies. The practical applications of phosphorimetry have been limited primarily as a result of the time and difficulty of sampling. Because analytical phosphorimetry is carried out a t low (e.g., 77 OK, in liquid nitrogen) temperatures (5), severe limitations concerning the choice of solvent and sample cell are imposed. The criteria (6) generally used to select a solvent for phosphorimetry have been solubility of the analyte, low luminescence background, and the formation of a clear glass. Unfortunately, few solvents are able t o meet these criteria a t liquid nitrogen temperature. In addition, the selected solvent almost always must be purified t o reduce trace concentrations of phosphorescent impurities. Long, thin-walled quartz tubes are most commonly used for sample cells (7), but such cells are of limited use with solvents which form highly cracked or snowed matrices, such as water, because the cells themselves are subject to strains caused by solvent expansion, which often results in shattering of the quartz sample tube. Solvents which are crystalline or severely cracked a t 77 OK have been used previously almost exclusively for qualitative Author to whom reprint requests should be sent. (1) R. J Keirs, R. D. Britt, and W. E. Wentworth, ANAL.CHEM., 29, 202 (1957). (2) C. A. Parker and C. G. Hatchard, Analyst, 87,664 (1962). (3) M. Zander, “The Application of Phosphorescence to the Analysis of Organic Compounds,” Academic Press, New York, N.Y., 1968. (4) J. D. Winefordner, P. A. S t . John, and W. J. McCarthy, “Fluorescence Assay in Biology and Medicine,” Volume 11, S. Udenfriend, Ed., Academic Press, New York, N.Y., 1969. ( 5 ) C. A. Parker, “Photoluminescence of Solutions,” Elsevier, New York, N.Y., 1968. (6) J. D. Winefordner and P. A. St. John, ANALCHEM.,35, 2211 (1963). (7) J. D. Winefordner and H. W. Latz, ihid., p 1517.
studies (8). Zweidinger and Winefordner (9) have recently reported the use of solvents forming snowed matrices for quantitative studies. Theoretical equations dealing with the optical inhomogeneities in snowed media have been derived and applied to the explanation of shapes of analytical curves of phosphorescence signal E S . analyte concentration. The use of solvents containing a high percentage of water was again restricted because of sample cell cracking. The use of internal standards for quantitative work in snowed matrices (IO) is difficult because phosphorescence intensity may be influenced by the extent of scattering of both exciting and emitted radiation. Scattering can be wavelength dependent under these conditions, because the effective particle size is small (21). In this manuscript, the use of a new, convenient microsample cell for quantitative studies in essentially aqueous solvent systems a t 77 OK is reported. Aqueous solvents are desirable in phosphorimetry because they are readily prepared in pure form, requiring n o additional lengthy purification procedures. Also, solution parameters, such as pH, which can greatly influence phosphorescence intensity ( I 2 ) , are welldefined and more easily adjusted. The sample cell proposed is a n open-ended quartz capillary tube. Quartz capillary tubing is available commercially, but not always of the highest quality, Trace amounts of metal impurities in the quartz can cause large phosphorescence background signals (13). A simple and effective procedure for greatly reducing the background, employing polarized exciting light will be described in detail. Analytical utility for phosphorimetry in aqueous solutions, using the capillary cell will be demonstrated by comparing analytical curves obtained for certain molecules in the capillary cell at 77 OK with analytical curves obtained for the same molecules at 77 OK in conventional sample cells. EXPERIMENTAL
Instrumentation. A n Aminco-Bowman spectrophotofluorometer with a n Aminco-Keirs phosphoroscope attachment, a 150-watt xenon arc lamp, and a potted R C A 1P28 photomultiplier tube (American Instrument Co., Silver Spring, Md.) were used for all studies. A n Aminco elliptical condensing system was employed to gather and focus exciting radiation. Polarization measurements were made using Glan-Thompson polarizers. Photomultiplier tube current was amplified and the signal measured with a low-noise nanoammeter ( 1 4 ) . Spectra were recorded with a n X-Y (8) Y. Kanda and R. Shimade, Spectrochim. Acta, 17, 279 (1961). (9) R. Zweidinger and J. D. Winefordner, ANAL.CHEM.,42, 639 (1970). (10) C. A. Parker and G. Hatchard, Tratis. Faraday Soc., 57, 1893 (1961). Cliem. Ztzt. (11) G. Kortiim, W. Braun, and G. Herzog, - Angew. Ed., 2, 333 (1963). (12) S. G. Schulman and J. D. Winefordner, Talanta, 17, 607 (1970). (13) C. A. Parker, “Photoluminescence of Solutions,” Elsevier, New York, N.Y., 1968,(a) p 235; (b) p 4498. (14) T. C. OHaver and J. D. Winefordner, J . Chem. Educ., 46, 241 (1969).
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plotter. A more detailed description of the instrumentation has been given previously (9). Preparation and Characteristics of Quartz Capillary Tubing. Synthetic high purity optical grade quartz capillary tubing, approximately 1-mm i.d. and 6-mm 0.d. (Quartz Scientific, Eastlake, Ohio) was cut into 10-in. lengths and cleaned using the recommended procedure (15). Both ends of the tube were ground smooth t o avoid scratching the quartz dewar flask when positioning the sample cell. To improve sample positioning the open-ended quartz tube was firmly fitted t o a n Aminco phosphorimetry sample tube holder using Teflon (Du Pont) tape. The inherent photoluminescence of the capillary tube was studied by recording room and low temperature spectra of both the prompt and long-lived luminescence components. Spectra were recorded with and without use of the GlanThompson polarizers. All possible combinations of simultaneous polarization of both exciting radiation and emitted radiation involving mutually orthogonal orientations of the polarizers were tried. Spectra were also obtained using only one polarizer a t a time. Reagents. Reagents used without further purification were: 2-thiouracil, sulfamethazine (Nutritional Biochemical Corp., Cleveland, Ohio); p-nitrophenol (Fisher Scientific Co., Fair Lawn, N. J.), and spectroquality methanol (Matheson, Coleman and Bell, East Rutherford, N.J.). Water used as a solvent was obtained directly from a commercial ion exchange column and was not further purified. Choice of Solvents for Quantitative Studies. Water, being one of the most convenient and available solvents, was the first choice for quantitative studies using the quartz capillary tube. Preliminary investigation of the phosphorescence spectrum of 2-thiouracil in aqueous solution indicated a drastic reduction of the phosphorescence signal compared with that obtained for 2-thiouracil in high purity ethanol solution (16). The spectral distribution of absorbed and emitted radiation was nearly the same as that obtained in 100% ethanol; however, the phosphorescence signal was reduced by nearly three orders of magnitude. The signal decrease is believed t o be the result of a complex matrix effect, and will be discussed in greater detail in the following sections. It was discovered that addition of small amounts of alcohol (ethanol or methanol) t o the aqueous solutions of 2-thiouracil both changed the physical appearance of the frozen matrix and increased the signal t o approximately the same level obtained in a clear rigid matrix employing ethanol which was purified by vacuum distillation. Frozen aqueous solutions are severely cracked but translucent, whereas frozen alcoholicaqueous solutions are completely snowed, even with as little as 10% VjV of alcohol. Both 95% ethanol and spectroquality methanol were mixed with water in varying proportions and tried as solvents. The nature of the matrix and the phosphorescence signals appeared t o be approximately the same for both, over a range of 50z VjV t o approximately 10 % VjV alcohol-water. The phosphorescence background signal, however, was much smaller for methanol-water than for ethanol-water mixtures, indicating spectrograde methanol is much more pure than 9 5 z ethanol. Therefore, the solvent system chosen for analytical studies was 10% VjV methanol-water. Also, neither component of the mixed solvent required further purification. Procedures. All luminescence background spectra of the quartz capillary tube are taken with the sample tube suspended in a n Aminco quartz window dewar flask (7). Long-lived luminescence spectra are obtained using the phosphoroscope attachment. Prompt luminescence spectra are obtained with the phosphoroscope attachment removed. The slit arrange(15) R. Zweidinger, L. B. Sanders, and J. D. Winefordner, Alia/. Chim.Acta, 47, 558 (1969). (16) L. B. Sanders, J. J. Cetorelli, and J. D. Winefordner, Talanta, 16,407 (1969).
238
0
600
Wavelength (nm)
Figure 1. Excitation and emission spectra of longlived component of quartz capillary sample tube Spectra 3, 4, 5, and 6 correspond to excitation at wavelengths 230, 250, 220, 280 nm, respectively. Full scale approximately 8 times level of dark current of PMT
ment used for all studies is 3,4,3,3,4,3 (all in mm, corresponding to approximately 17 and 22 nm spectral half-band pass). Spectra are not corrected ( 4 ) for instrumental response. A 0.5-second time constant is used in the nanoammeter. Stock solutions which were 10-3M in 10% VjV methanolwater were prepared, Analytical curves were prepared from successive dilutions of the stock solutions. All determinations were made in triplicate. The limit of detection is defined here as that concentration which gives a signal just distinguishable from the background. Signal-to-noise ratio using this definition is about 4 t o 1. Introduction of sample solution into the capillary tube is much simpler and more convenient than previously reported procedures ( 7 ) . Because the capillary tube is open-ended, it is necessary only t o dip the tube into the sample solution. The surface tension of the liquid determines quite reproducibly the amount of sample retained in the capillary tube. For the solvent system used, a sufficient volume of sample is retained t o provide for an even distribution of sample throughout the height of the exciting radiation beam. After the outside of the tube is wiped dry, the sample is immersed slowly (requires about 10 seconds) into the liquid nitrogen containing dewar flask, while holding the index finger firmly on the top of the capillary to prevent bubbling from forcing the solution up the tube. After a phosphorescence reading is taken, the sample in the cell is thawed by blowing hoi air over it, and the sample is removed by flushing with nitrogen. Between determinations, the capillary cell is cleaned by rinsing several times with the solvent. After an analytical curve is determined, the tube is left standing in concentrated H N 0 3to remove any adsorbed material (15). RESULTS AND DISCUSSION Spectral Characteristics of Quartz Capillary Tube. Luminescence excitation and emission spectra of the low temperature, long-lived components of the quartz capillary tube are given in Figure 1. The spectra were obtained with unpolarized excitation light and unpolarized luminescence. Two distinct maxima occur in both the excitation and emission spectra. A broad emission band centered at 470 nm is observed when the tube js excited a t 212 nm. A somewhat narrower emission maximum appears at 400 nm when the excitation wavelength is 240 nm. Three much smaller maxima appear in the excitation spectra between 300 and 500 nm. Excitation at 335
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am
300
403 Wavelength (nm)
\
600
Concentrotton (pg/rnl)
600
Figure 3. Experimental analytical working curves for 2thiouracil ( A ) ; p-nitrophenol ( B ) ; sulfamethazine (C); in 9 : l V/V methanol-water at 77 O K ; phosphorescence signal in amperes 6s.concentration in pg/ml
Figure 2. Excitation and emission spectra of longlived component of quartz capillary sample tube, using polarized radiation; orientation of polarizers with respect to vertical axis of cell as follows: 1. 2.
Excitation
Emission
I
/I
I1
I1
3. I I I 4. II No polarizer 5. I No polarizer 6. I1 7. No polarizer I 8. No polarizer I/ Spectra 1 thru 6 were obtained with a gain of 5-fold; fullscale approximately equal to twice level of dark current of PMT. Spectra 7 and 8 were obtained with a gain of unity
n m gives rise to an emission peak at 400 nm. The existence of two distinct peaks in the excitation and emission spectra is believed to be the result of trace impurities of at least two different metals. Parker (13b) reported the same observation and has attributed the longer wavelength emission to trace amounts of copper and the blue emission to small amounts of aluminum. Assignment of any of the maxima in Figure 1 as scattered light is unlikely because excitation and emission radiation are resolved in time by the rotating can phosphoroscope. Prompt luminescence from the capillary tube is not observed under these conditions. Prompt luminescence spectra a t both low and room temperature were obtained for the tube by removing the phosphoroscope attachment. Possible interference by scattered radiation was minimized by recording several scans of the luminescence spectrum a t different excitation wavelengths and superimposing the emission spectra. Only those peaks common to all the scans were interpreted as prompt luminescence. Low temperature prompt luminescence peaks were observed a t 295 nm, 392 nm, 544 nm, and 572 n m when excited at 240 nm, the latter two emission peaks could also be observed by exciting at 340 and 365 nm, respectively. Room temperature prompt luminescence peaks were observed a t 400 n m and 540 n m when excited a t 240 and 230 nm, respectively. Longlived luminescence did not interfere with prompt luminescence spectra because the former is at least 3 orders of magnitude less intense under the experimental conditions employed. Because the long-lived sample tube luminescence signal was nearly an order of magnitude greater than the dark current of the photomultiplier tube, an attempt was made to reduce the background by using Polarized radiation. Luminescence excitation and emission spectra of the sample tube using plane
polarized radiation are given in Figure 2. I t can be seen that the lowest background was obtained when both polarizers were used. F o r quantitative work, however, use of both polarizers is undesirable because each polarizer has a low transmittance, and the loss of sensitivity is compounded. The background resulting when a polarizer was used only in the excitation beam is slightly higher than that obtained with two polarizers but still approximately 100 times lower than the background obtained without the use of polarizers. A significant increase in the luminescence background signal is observed when a polarizer is used only in the emission beam. The wavelengths of peak luminescence of the excitation and emission spectra are also shifted when a polarizer is used only in the emission beam; the reasons for these phenomena are not completely understood. It can also be seen from Figure 2 that the luminescence excitation and emission background is always smaller when the cell is excited with radiation polarized in the plane perpendicular to the tube. F o r these reasons, quantitative studies were undertaken with excitation radiation polarized perpendicularly and unpolarized emitted radiation. Solvent and Matrix Considerations. Although the use of water as a solvent for phosphorimetry previously was not possible because of the shattering of conventional sample cells, with the improved sample tube aqueous solutions gave signals 3 orders of magnitude smaller than that obtained for clear glasses. Similar results have been observed by other workers (17). It is believed that the nature of the matrix, i.e., extent of cracking, is the reason for the signal decreases. Zweidinger and Winefordner (9) induced cracking in propylene glycol-water solutions, by introducing a small plug of quartz wool just above the optical path. In their paper, equations were derived predicting that a t low concentrations, the signal obtained in snowed matrices should be twice that observed in clear glasses. The experimentally observed value measured by Zweidinger and Winefordner (9) was 1.7; equations were derived assuming the matrix to be a diffusely reflecting media. Presumably the enhancement of signal was due to multiple reflections of exciting radiation within the matrix. Conversely, in pure water solutions, the degree of cracking is smaller, the particle size is larger (9), and the resulting lumi(17) A . Hornig, Baird Atomic Inc., Bedford, Mass., personal communication, 1971.
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Table I. Phosphorescence Characteristics of Molecules Studied Slope of Compound linear portion Limit of detection, pg/ml p-Nitrophenol0 0.88 7 x 10-3 Sulfamethazinea 1.01 6 x 10-3 2-ThiouraciP 0.81 7 x 10-4 Solvent 10% (V/V) methanol; water, all solutions formed snowed samples. * Volume of sample retained in capillary tube calculated as 17.8 11. c Solvent 10% (VjV) methanol; water with 1 by volume saturated NaOH added.
nescence signal is correspondingly smaller. In this case, specular reflection of excitation radiation is larger (18) which may direct the excitation beam away from the matrix to cause the drastic reduction in signal. Another possible explanation for the reduced signal is the formation of inhomogeneous concentration gradients caused by molecular aggregation in aqueous solution (17). The degree of cracking, however, can be greatly enhanced by the addition of small amounts of alcohol to the water used as solvent. This obviates the problems encountered with the nature of the matrix, and makes aqueous solutions analytically useful in phosphorimetric analysis. Analytical Studies. To demonstrate the analytical usefulness of the capillary cell and aqueous solvents, phosphorescence analytical curves were obtained for three model compounds (see Figure 3 ) . The range of linearity compares favorably with that obtained by Zweidinger and Winefordner ( 9 ) in similar snowed matrices. Slopes of the working curves and limits of detection are given in Table I. Slopes for p-nitrophenol and 2-thiouracil analytical curves are smaller than the normally accepted value of unity. Theoretical equations predicting the shapes of analytical curves for snowed matrix experiments, assuming a diffuse reflectance model, have been derived (9). Consider Equation 1 derived by Zweidinger and Winefordner (9).
Ip
=
F
29,IOkb[---1
-
1
7
1+2sb I p is the phosphorescence intensity, the quantum yield, I o intensity of exciting radiation, k is the fraction of light absorbed (2.303 &) per cm, b the path length, s the fraction of radiation scattered per average path length o r the scattering coefficient of the matrix. A t low analyte concentrations, two postulations may be made concerning Equation 1. First, if the product sb is considerably less than 0.5, the analytical signal obtained is actually enhanced over that observed in clear glasses. As sb approaches 0.5, the signal obtained becomes equivalent to that for a clear glass. The second postulate is that the slope of a log I p US. log C plot will be unity. The latter postulate has been (9) verified for sulfanilamide and also oxythiamine in a snowed matrix (4:l VjV isooctaneethanol). However,, a negative deviation from a slope of (18) W. W. Wendlandt and J. G. Hecht, “Reflectance Spectroscopy,” Interscience, New Ynrk, N.Y., 1966.
240
Number of gramsb at limit of detection 1.24 x 10-10
Concentration range of near linearity 104 104 104
1.07 X 10-10 1.24 x 10-11
unity was obtained for some molecules in mixed aqueous solutions. Two of the three compounds used in this study also showed the same behavior in mixed aqueous solutions. Because some compounds give slopes of unity, sulfamethazine in this study, in aqueous mixed solvents, a relationship between the overall scattering coefficient and the analyte concentration used may exist. We are currently investigating this phenomenon in more detail. The limits of detection measured with samples in the capillary cell compare favorably with those previously reported in clear rigid glasses. The limit of detection for 2-thiouracil is about 1 order of magnitude lower than that previously reported (16); limits for p-nitrophenol and sulfamethazine are about 1 order of magnitude higher than those previously reported (19). Comparison of limits of detection is difficult because instrumental parameters and the definition of limit of detection used in this study differ considerably from those of other workers (19). A quite conservative definition for the detection limit was utilized in these studies. Because the volume of sample is quite small, about 20 pl, the total number of grams necessary for analysis is in the picogram range. This is especially convenient when the amount of sample available is extremely small. Such cases are often encountered in clinical chemistry. The analytical utility of phosphorimetry has been extended to include aqueous solutions for the first time. Sample preparation and handling have been substantially simplified, and the sensitivity of the technique has been retained. Further work including acquisition and testing of higher quality capillary tubes, which hopefully will eliminate the need for using polarized radiation is in progress. Attempts to improve cell positioning will also be made; the relative standard deviations using the present capillary cell are typically 10-12 % for the molecules studied over the entire concentration range. The precision was limited by the error of cell positioning; future studies will include use of a rotating sample capillary cell to randomize sample cell positioning errors (9). RECEIVED for review June 11, 1971. Accepted September 20, 1971. Research was carried out as part of a study on the phosphorimetric analysis of drugs in blood and urine supported by a U.S. Public Health Service Grant, GM-11373-09. _
_
_
~
-
(19) W. J. McCarthy and J. D. Winefordner, “FluorescenceTheory, Instrumentation, and Practice,” G. G. Guilbault, Ed., Marcel Dekker, New York, N.Y., 1967.
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