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Anal. Chem. lQ84, 56,836-837
Instrumental Modification for Room-Temperature Phosphorescence David L. McAleese and R. Bruce Dunlap* Department of Chemistry, University of South Carolina, Columbia, South Carolina 29208 Room-temperature phosphorescence (RTP) is a rapidly developing luminescence technique for the quantitation of important organic and biological compounds. Molecules exhibit the phenomenon when adsorbed on a suitable solid support. Cellulose filter papers are credited as the most applicable R T P supports to date (1). Typically, a few microliters of analyte solution is applied to circles, strips, or rolls of filter paper. Since moisture quenches phosphorescence emission, the samples are dried prior to analysis in a phosphorimeter. A problem which exists with the technique, however, is that the sample sizes previously reported (1-17) are larger than the excitation beam in conventional phosphorimeters such as the Aminco-Bowman spectrophotofluorometer. Thus, migration of phosphors outside the illumination area on the support causes a loss in phosphorescence sensitivity. The extent of migration depends on the chromatographic behavior of the phosphor on the cellulose paper chosen. For example, Vo-Dinh et al. (2) scanned the sample spots with a continuous filter paper device and observed that the phosphorescence emission profiles were varied in shape. Two maxima a t the borders of the emission curve were observed for p-aminobenzoic acid indicating that a large quantity of the phosphor eluted near the edge of the spot. Yen-Bower and Winefordner (3)recorded emission profiles noting that all investigated ionic molecules exhibited a major peak with shoulders or in some cases two distinct peaks as for carbazole. Partial illumination of the samples has also accounted for a considerable lack of precision in the RTP technique. Since an uneven distribution of phosphors on the supports is produced due to chromatography, partial illumination of the samples complicates the task of illuminating the same amount of analyte from sample to sample. It seemed logical to us then that total front surface illumination of the samples could improve both the sensitivity and precision of phosphorescence measurements. In this investigation, we modified the Aminco-Bowman instrument and reduced the size of the filter paper so that complete front surface illumination could be accomplished.
EXPERIMENTAL SECTION Apparatus. Phosphorescence intensity measurements and excitation/emission spectra were obtained on an Amincc-Bowman spectrophotofluorometer equipped with a 150-Wxenon arc light source, a phosphoroscope, a laboratory-constructedsample holder (I), and a 1P21 photomultiplier tube detector. Chemicals. Reagent grade p-aminobenzoic acid (PABA), 4-biphenylcarboxylic acid (4BPCA), and sodium citrate were utilized without any further purification. Solutions of PABA and 4BPCA were refrigerated in the dark to prevent degradation. Sample Preparation. Two microliters of an ethanol solution containing the phosphor was applied on either a 6.4 mm diameter circle of Whatman No. 1 qualitative filter paper or a 3.2 mm diameter circle of Whatman No. 3 MM chromatography paper. After desiccation of the samples for 30 min inside a glovebag attached to the sample compartment of the instrument, 2 WLof a 1 M aqueous sodium citrate solution (pH 7) was applied to the filter paper supports. It has been shown that the presence of sodium citrate eliminates oxygen and moisture quenching of the RTP emission of dried samples (4). The samples were analyzed after a final 2-h desiccation period. Excitation Monochromator Modification. The AmincoBowman spectrophotofluorometer contains a Czerny-Turner type excitation monochromator. The collimating mirror closest to the
light source was shifted 3.5 cm toward the plane grating. This decreased the distance between the center of the mirror and grating from 17 cm to 13.5 cm. The mirror was anchored in place with two of the four screws provided. The small black plates partially covering the left side of the mirror and grating were replaced with larger black shields to eliminate white light and second-order light interferences generated by the mirror displacement. The shields covered approximately one-third of the area on the mirror and grating. No problems were encountered in the spectral calibration of the excitation monochromator.
RESULTS AND DISCUSSION The Aminco-Bowman spectrophotofluorometer contains a xenon lamp which produces an arc 3.0 mm in length. The Czerny-Turner type excitation monochromator focuses a monochromatic image of the lamp in the center of the sample compartment. However, the strips, rolls, and 6.4 mm diameter circles of filter paper previously used for RTP are longer than the 3.0 mm vertical image of the excitation beam. These supports are also wider than the focused image when small slits are inserted in the path of the excitation beam. Analyte solutions of 2 pL or more are sufficient to cover a 6.4-mm Whatman No. 1 filter paper circle completely, so migration of phosphors outside the illumination area on the support occurs. The application of 1-pL solutions to filter paper still produces a larger (4 mm) circle. In order to irradiate the entire surface area of the sample, the vertical image of the excitation beam incident on the sample was increased to a homogeneous 4 mm beam, the support diameter was reduced to 3.2 mm and excitation slits were removed. The ionic compounds p-aminobenzoic acid (PABA) and 4-biphenylcarboxylic acid (4BPCA) are strong phosphors and would be expected to elute on filter paper to different extents. The chromatographic behavior of both compounds was tested on strips of Whatman No. 3 MM paper with the same solvents used in the phosphorescence experiments. Both compounds eluted with the ethanol solvent front as observed under ultraviolet light. However, PABA eluted near the solvent front of the aqueous sodium citrate solution while 4BPCA was highly retained. PABA would, therefore, migrate toward the edge of the RTP paper circle to a greater extent than 4BPCA. A comparison of phosphorescence intensities and precision is shown for PABA and 4BPCA as the fraction of total sample surface area exposed to the excitation light was increased (Table I). The illumination area percentages were calculated on the basis of the placement of the excitation beam in the exact center of the supports. Since this alignment was difficult to attain and verify experimentally, the first three area percentages were regarded as only approximate. Any deviation of the beam from the center of the supports reduced the illumination area. The first sample had the smallest fractional area irradiated and PABA exhibited a 45.0% lower intensity than 4BPCA. The relative standard deviation of 10 measurements of PABA was 2.5 times greater than 4BPCA. With about a 2-fold increase in illumination area for the second sample, the PABA phosphorescence exceeded that of 4BPCA by 12.5%. The relative standard deviation of the PABA samples was reduced by half. Most of the sample area was irradiated in the third sample as the PABA intensity increased to 17.8% over 4BPCA. The precision of both compounds was improved to about 3%. Finally, the entire surface area was exposed in the fourth sample and the PABA phosphorescence increased further to 26.0% over 4BPCA. Both relative
0 1984 American Chemical Society 0003-2700/84/0356-0836$01.50/0
Anal. Chem. 1984, 56,837-839
Table I. Effects of Partial Sample Illumination on RTP Measurements
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4-biphenylcarboxylic acid sample p-aminobenzoic acid diameter. excitation mirror % area % mm slita position illuminated RI % R S D C BDW,d nm R I b RSDC BDW,dnm 6.4 0.5 mm standard 30 9.85 11.9 45 17.9 4.74 40 6.4 open standard 57 100 5.85 51 88.9 4.79 47 3.2 open standard 98 69.6 2.85 45 59.1 3.16 40 3.2 open shifted 100 53.8 1.32 45 42.7 1.06 40 a Includes open emission slit and 5-mm photomultiplier tube slit. Average relative intensity of 1 0 samples, each containing 100 ng of phosphor. Maximum excitation/emission wavelengths are as follows: PABA, 2861422 nm; 4BPCA, Relative standard deviation of 10 samples. 289/482 nm. Excitation spectral bandwidths. standard deviations were reduced significantly to excellent values of 1.32% and 1.06%. The PABA phosphorescence intensity increased a total of 71.0% with respect to 4BPCA as the fractional area illuminated was increased. This indicated that more PABA molecules eluted toward the edge of the paper circle than 4BPCA and that a loss in phosphorescence sensitivity was incurred for PABA samples incompletely irradiated. The intensities of both phosphors decreased from the second to fourth samples with increasing illumination area percentage. The reason for the decrease from sample 2 to sample 3 was not entirely clear, but the reduction was accompanied by a similar loss (35%) in background emission. The decrease from sample 3 to sample 4 resulted from the mirror displacement in the excitation monochromator. The optical modification broadened the vertical band of excitation light to a greater length than the support diameter. Therefore, the power of the excitation light impinging on sample 4 was less than that on sample 3. When the mirror was displaced the distance required for complete illumination of the 6.4 mm circles, the power of the excitation light was too small for analytical work. It was necessary to cover most of the mirror and grating to eliminate white light and second-order light interferences. This decreased the throughput of the excitation monochromator to a point that instrumental noise contributed significantly to the phosphorescence emission signals. R T P excitation and emission spectra were recorded for the different PABA and 4BPCA samples in Table I. With the optical modification and reduced filter paper support size (sample 4), the excitation spectral bandwidths were found to be identical with the 6.4-mm samples with a 0.5-mm excitation slit and no modification of the monochromator (sample 1). Since it was visually determined that 0.5-mm slits allowed total horizontal surface illumination of the 3.2-mm samples, the use of 0.5-mm slits or larger was unnecessary for R T P measurements. Slits smaller than 0.5 mm did not reduce the spectral bandwidths further and would have only served to introduce sensitivity and precision problems associated with incomplete surface illumination of the samples. The emission spectral bandwidths of sample 4 were also identical with the bandwidths of sample 1 with a 0.5-mm emission slit. The emission slits were therefore excluded to maximize phosphorescence collection efficiency. A direct comparison of the quantitative data for samples 1 and 4 of Table I indicated that sensitivity was gained a t no
expense of spectral broadening and, thus, with no loss of spectral resolution. The PABA and 4BPCA intensities increased 5.46-fold and 2.39-fold, respectively, while the relative standard deviations decreased 9.02- and 4.47-fold. The increased sensitivities manifested good experimental detection limits for the two phosphors. From the least-squares analyses of the log intensity vs. log concentration working curves, the calculated limits of detection for PABA and 4BPCA were 12.9 pg and 31.7 pg, respectively, at a signal-to-noiseratio of 2. The PABA detection limit was 7.8- to 780-fold lower than those previously reported on solid supports including paper (5-8). These studies clearly demonstrate the importance of total front surface illumination of samples for improved sensitivity and precision in the R T P technique. Registry No. PABA, 150-13-0;4BPCA, 51317-27-2.
LITERATURE CITED (1) Parker, R. T.; Freedlander, R. S.; Dunlap, R. B. Anal. Chlm. Acta 1980, 120, 1-17. (2) Vc-Dinh, T.; Waiden, G. L.; Winefordner, J. D.Anal. Chem. 1977, 49, 1126-1130. (3) Yen-Bower, E. L.; Winefordner, J. D. Appl. Spectrosc. 1979, 33, 9-12. (4) McAleese, D. L.; Freedlander, R. S.;bunlap, R. B. Anal. Chem. 1980, 5 2 , 2443-2444. (5) Ford, C. D.; Hurtubise, R. J. Anal. Chem. 1979, 5 1 , 659-663. (6) Von Wandruszka, R. M. A.; Hurtubise, R. J. Anal. Chem. 1976, 48, 1784- 1788. (7) Vo-Dinh, T.; Yen, E. L.; Winefordner, J. D. Anal. Chem. 1976, 4 8 , 1186-1 188. ( 8 ) Welions, S . L.; Paynter, R. A.; Winefordner, J. D. Spectrochlm. Acta, PartA 1974, 30A, 2133-2140. (9) Parker, R. T.; Freedlander, R. S.; DuniaD, R. B. Anal. Chim. Acta 1980, 119, 189-205. (10) Ward, J. L.; Bateh, R. P.; Winefordner, J. D. Ana/yst (London) 1982, io7 . - . , 335-338 - - - - - -. (11) Vc-Dinh, T.; Gammage, R. B.; Martinez, P. R. Anal. Chim. Acta 1980, 118, 313-323. (12) Dalterio, R. A.; Hurtubise, R. J. Anal. Chem. 1982, 5 4 , 224-228. (13) Bateh, R. P.; Winefordner, J. D. Anal. Left. 1982, 15 (B4), 373-383. (14) Vo-Dinh, T.; Gammage, R. B.; Martinez, P. R. Anal. Chem. 1981, 5 3 , 253-258. (15) Warren, M. W.; Avery, J. P.; Maimstadt, H. V. Anal. Chem. 1982, 54, 1853-1858. (16) Ramasamy, S. M.; Hurtubise, R. J. Anal. Chem. 1982, 5 4 , 1642- 1644. (17) Bateh, R. P.; Winefordner, J. D. J. Pharm. Sci. 1983, 72, 559-560.
RECEIVED for review July 18, 1983. Accepted December 7 , 1983. These investigations were supported by NIH Grant CA 15645 from the National Cancer Institute. R. Bruce Dunlap is the recipient of a Faculty Research Award (FRA-144) from the American Cancer Society.
High-speed Switching of Liquid Chromatographic Injection Valves Mack C. Harvey* a n d Stanley D. S t e a r n s Valco Instruments Co., Inc., P. 0. Box 55603, Houston, Texas 77255 When a sample is injected onto an HPLC column with an injection valve, the flow of the mobile phase from the pump
to the HPLC column is interrupted as the valve moves from one position to the other. Since most HPLC pumps are of
0003-2700/84/0356-0837$01.50/00 1984 American Chemical Society
