Heavy-Atom Effect on Room Temperature Phosphorimetry Tuan Vo-Dinh, Esther Lue Yen, and James D. Winefordner" Department of Chemistry, University of Florida, Gainesville, Fla. 326 1 1
The use of sodium iodide as an external heavy atom perturber in the room temperature phosphorescence method has been investigated lor a wide varlety of compounds of biological and pharmaceutical Interest. In most cases, sodium iodide has been found to be very efficient in increasing the phosphorescence quantum yield and thus improved significantly the limits of detection of this rapid and simple method of molecular analysis.
Phosphorimetry has been developed into a sensitive spectrochemical method of analysis. Various recent developments and applications, especially in clinical chemistry, have been reviewed ( I , 2). In most phosphorimetric studies, the analysis has to be performed using either low temperature rigid matrices, such as organic glasses a t liquid nitrogen temperature or carefully-degassed solutions in order to minimize collisional triplet-quenching. The major analytical disadvantages of phosphorimetry are related to the need of cryogenic equipment or time-consuming degassing procedures, making this technique less-widely used than fluorimetry. Very recently, i t has been reported that intense phosphorescence was observed at room temperature of various salts of polynuclear organic compounds when adsorbed on filter paper, silica gel, alumina, and polycellulose (3-5). Room Temperature Phosphorimetry (RTP) has been thus suggested as a quantitative tative analytical method (6). Also, the external heavy atom effect on phosphorescence measured at room temperature has been reported with 2halonaphthalene sulfonate and some of its derivatives (7). Since the first report of solvent heavy-atom effect was given in 1952 by Kasha ( 8 ) ,who observed an increase in the oscilSo transition of l-chloronaphlator strength of the T I thalene by mixing it with ethyl iodide, it has been confirmed that the presence of heavy atoms, either as substituents (internal heavy-atom effect) or in the environment (external heavy-atom effect) can enhance significantly the process of intersystem crossing (9) and, therefore, the phosphorescence emission. In order to investigate the possibilities and improvements of this rapid and simple method of analysis, this work reports the external heavy-atom effect on phosphorescence a t room temperature. The samples selected for our investigation consist of a wide variety of compounds of biological and pharmaceutical interest.
-
EXPERIMENTAL Apparatus. The experimental set-up used in this study was previously described in detail (6).Phosphorescence measurements were performed with an Aminco-Bowman spectrophotofluorimeter with an Aminco-Keirs phosphoroscope attachment (American Instrument Co., Silver Spring, Md.). A 150-W xenon arc lamp was used as the excitation light source and the signal detected with a 1P21 photomultiplier tube with S-4 spectral response. Large monochromators slit widths (5 mm) were used for photometric detection limits, whereas higher spectral resolution was used for recording the spectra (1-mm monochromator slit width corresponding to 5-nm spectral resolution). The spectra were recorded on an x-y recorder (MFE Corp., Salem, N.H.).
Reagents. Eaton Dikeman 613 filter paper was used as support for all analytical investigations. Adenine, barbituric acid, guanine, 61186
ANALYTICAL CHEMISTRY, VOL. 48, NO. 8, JULY 1976
chloropurine, 6-methylmercaptopurine,sulfanilamide (Nutritional Biochemicals Corp., Cleveland, Ohio); L-(-)-tryptophan (Eastman Organic Chemicals, Rochester,N.Y.);p-aminobenzoicacid, salicyclic acid (Fisher Scientific Co., Fair Lawn, N.J.); cocaine hydrochloride (Applied Science Labs, Inc., State College, Pa.) were all used as received. Sodium hydroxide (Matheson Coleman and Bell, Norwood, Ohio) and sodium iodide (J. T. Baker Chemical Co., Phillipsburg, N.J.) were used to prepare the solvent. Procedure. Sample solutions were spotted on 0.25411. diameter filter paper circles and dried for about 30 min by infrared lamp heating. This technique has been shown to be more efficient than other methods using desiccators, blowers, or ovens. After the drying procedure, the samples were directly mounted on a simply designed holder (6). Since the phosphorescence emission is very sensitive to humidity, the sample compartment was continuously flushed with warm dried air. During all measurements, the temperature in the sample compartment was about 60 "C. RESULTS AND DISCUSSIONS Spectral Studies. The room temperature phosphorescence spectra of tryptophan adsorbed on filter paper from a 1 M NaOH aqueous solution (concentration = 6.6 X M) and a 1 M NaOH-1 M NaI solution (concentration = 7.3 x M) are shown in Figure 1. The presence of NaI greatly increases the observed phosphorescence signal; these two spectra in Figure 1 were recorded with different sensitivity scales. Obviously the heavy-atom enhancement technique affords considerable advantage in quantitative analysis since the limits of detection can be decreased, in some cases, by several orders of magnitude. Previously heavy metal ions, such as Hg2+ and Ag+, have been found to affect the phosphorescence of nucleosides and tryptophan a t 77 K ( 1 0 , l l ) .In the case of tryptophan, Hg2+ shortened the phosphorescence lifetime but also decreased the emission intensity, suggesting a quenching mechanism which might occur at the singlet levels (12).Ag+ metal ion has been found to increase substantially the phosphorescence intensity but, on the other hand, completely altered the vibrational structure. In the case of NaI used in R T P method, the emission intensity has been increased and the basic vibronic features were not significantly altered by the heavyatom perturbation; this indicated that the presence of NaI did not significantly change the nature of the emitting species and the radiative process. However, because of the low spectral resolution used in the present work, further work is needed prior to generalizations concerning the heavy-atom effect observed and the specific mechanism by which it occurs. In all measurements, the signals of the reference blanks showed an intensity increase of almost three times with NaI indicating that the paper emission is also affected by heavy-atom perturbation. Drying Conditions. The rigidity of the emitting species adsorbed on the paper support, and therefore the dryness of the sample is the main parameter affecting the phosphorescence emission intensity. The presence of moisture in the air can greatly reduce the phosphoresence intensity. After the paper sample has been exposed to the atmosphere and the moisture contained therein by removing the warm dry air supply, the phosphorescence signal decreased drastically. On the other hand, drying time has a reverse effect by increasing the phosphorescence intensity (Figure 2). Since the solutions contained a large excess of NaOH, in the process of drying the
'
t
n3
r
300
400
500
600
10
20
h(nrn)
30
40
50
TIME (min)
Figure 1. Tryptophan phosphorescence and the effect of Nal
Figure 2. Effect of drying time on room temperature phosphorescence intensity (2 X mol I.-' tryptophan in 1 M Na0H:l M Nal solution)
(1) Tryptophan in I M NaOH (c = 6.6 X mol I.-'). (2) Blank phosphorescence (paper with 1 M NaOH). (3)Tryptophan in 1 M Na0H:l M Nal ( c = 7 . 3 X mol I.-'), scale X 10. (4) Blank phosphorescence (paperwith 1 M Na0H:l M Nal), scale X 10. (5)Tryptophan phosphorescence at 77 K (from Ref. 74)
Curve 1. Phosphorescence intensity vs. drying time. Curve 2. Phosphorescence intensity vs. time after drying air has been removed.
(arbitrary scale)
sample on the paper support, the sample might become incorporated into a NaOH-Na2C03 matrix because of the presence of C02 in the drying air stream. The effect of Na2C03 as well as of various other experimental parameters on the phosphorescence intensity is of great interest and is now under investigation. The noteworthy feature is that the presence of oxygen (a known efficient triplet quencher in liquid solution a t room temperature) has no effect on the phosphorescence signal of dry samples observed by RTP. Even in polymer samples where large polyatomic molecules are immobilized in plastics, smaller molecules such as 0 2 can diffuse at sufficiently high rates to quench efficiently the phosphorescent triplets (13). All the measurements were performed after about 20 min, at which time the phosphorescence emission signal did not show any further increase in intensity. Analytical Curves. The analytical curve of tryptophan adsorbed on filter paper from a 1 M NaOH-1 M NaI (1:l) aqueous solution is reported in Figure 3. The slope of the log-lcg analytical curve is close to unity, and the range of
16
10-
rd 4
$63
CONCENTRATION(molar)
Figure 3. Room temperature phosphorescence analytical curve for tryptophan in 1 M Na0H:l M Nal solution adsorbed on filter paper at 60 O C
Table I. Phosphorimetric Limits of Detection (L.O.D.) for Various Compounds of Biological Importance AbsoluteC L.O.D.
Limits of detection"
Compound Adenine Guanine 6-Chloropurine 6-Methylmercaptopurine I,-(-)-Tryptophan Sulfanilamide Cocaine hydrochloride Para-aminobenzoic acid Salicylic acid Barbituric acid
Excitation wavelength, nm
Emission wavelength, nm
in NaOH, mol 1.-l
290 280 290 290
470 450 460 458
3.5 x 10-4 4.0 x 10-5 1.5 X lo-?
290 280 285
448 427 460
280
430
in NaOH:NaI, mol 1.-' 6.0 X 3.0 x 10-5 4.0 x 10-5 2.0 x 10-7
IY'*l
6.0 X 5.0 X
1.5 x 10-7 3.0 X loe6
40.0 1.7
4.5 x 10-5
3.0 x 10-5
1.5
7.0 x 10-7
9.0
10-5
x
10-8
Ip
1.6 12.0 1.0 0.8
12.0
ICng 2cng 6.8 4.1 264. 23. 31.
0.12
31. 0.17
6.1 0.15 4.3 2.6 76. 51. 0.48 0.062
1.0 x 10-6 4.0 2.8 0.69 5.0 x 10-5 1.4 45. 32. Absolute limits of optical detection, See text for definitions. a Defined as that concentration resulting in a signal-to-noiseratio of 2. calculated for 5 - ~ sample 1 solutions, ICin NaOH solution, 2c in Na0H:NaI solution.
320 290
470 455
4.0 7.0
X
x 10-5
ANALYTICAL CHEMISTRY, VOL. 48, NO. 8, JULY 1976
1187
linearity is fairly large (-lo3). Compared with data of tryptophan in a solvent without NaI, the analytical linearity range has been increased by a t least an order of magnitude because of the decrease of the detection limit at the low concentrations range. Because it was observed that the compounds could be gradually decomposed by the strong basic solvent, all the measurements were performed using freshly-prepared samples. Limits of Phosphorimetric Detection. Table I reports the limits of phosphorimetric detection of a wide variety of compounds of biological importance. The sodium iodide heavy-atom enhancement factor IpN"'/Ip is defined as the ratio of the molar phosphorescence signal in 1 M NaOH-1 M NaI (1:l) solution to that in 1M NaOH aqueous solution, respectively, and was deduced from the following equation:
where ip*, ,'?i c p ' are, respectively, the phosphorescence signals (in A) of the sample, S, the reference blank, R, and the concentration (mol l.-I) of the substance, S, under investigation in a solution of NaOH-NaI; and the other parameters correspond to those items for only NaOH solution (no NaI). Except for 6-methylmercaptopurine, the heavy-atom enhancement factor was found for most compounds to be greater than unity. It ranged from 1.0 for 6-chloropurine to 12 for guanine, and 40 for tryptophan. The data show that the heavy-atom effect on room temperature phosphorescence varies appreciably with the molecular structure of the compounds and the nature of the excited states involved. Advantages of R T P f o r Analysis. An obvious advantage of R T P technique using paper for analytical purposes, besides its simplicity, is the small amount of sample which is required for every measurement; i.e., only 5 ~1 is usually needed to prepare a sample, whereas low temperature phosphorimetry usually requires 100 ~1 to 1ml and conventional fluorimetry requires as much as 4 ml. Therefore, the absolute sensitivity (in ng) of R T P is in some cases much higher than the con-
ventional techniques; for tryptophan, an absolute limit of detection of 0.15 ng can be achieved. The significant enhancement of tryptophan phosphorescence is of special interest in protein analysis since most proteins have been found to emit via their tryptophan residues. Para-aminobenzoic (PABA) acid which has a very low limit of detection (9 X mol l.-l) is also of medical interest because it has been used to raise blood salicylate level, or in the laboratory as a sulfonamide antagonist, or more commonly as a sunscreen agent (14). The absolute limit of phosphorimetric detection of PABA is 0.06 ng. This study has shown that the external heavy-atom effect on room temperature phosphorescence is applicable to a wide variety of compounds of biological importance. In some cases, the sensitivity of phosphorimetric detection is increased by several orders of magnitude. It is therefore suggested that the use of the external heavy-atom effect be applied to improve the sensitivity of room temperature phosphorimetric method of analysis. This technique, which is a rapid, simple method of analysis, would be quite suitable for clinical analysis. LITERATURE CITED (1) C. M. O'Donnell and J. D. Winefordner, Ciin. Chem., ( Winston-Salem,N.C.), 21, 285 (1975). (2) J. J. Aaron and J. D. Winefordner, Talanfa, 22, 707 (1975). (3) E. M. Schulman and C. Walling, Science, 178, 53 (1972). (4) E. M. Schulman and C. Walling, J. fhys. Chem., 77, 902 (1973). (5) P. G. Seybold, R. K.Sorrel1 and Schuffert, 165th National Meeting, American Chimical Society, Dallas, Texas, April 13, 1973. (6) S. L. Wellons, R. A. Paynter, and J. D. Winefordner, Spectrochim. Acta, fartA, 30, 2133 (1974). (7) P. G. Seybold and W. White, Anal. Chem., 47, 1199 (1975). (8) M. Kasha, J. Chem. fhys., 20, 71 (1952). (9) T. Medinger and F. Wllkinson, Trans. Farad. Soc., 61, 620 (1965). (10) R. F. Chen, Anal. Biochem. Biophys., 144, 552 (1971). (11) G. D. Boutilier, C. M. O'Donnell, and R. 0. Rahn, Anal. Chem., 46, 1508 (1974). (12) R . F. Chen, Amhco Fiuorescence News/., 9, No. 2 and 3, 9 (1975). (13) B. A. Baldwin and H. W. Otfen, J. Chem. fhys., 49, 2933 (1968). (14) The Merck Index, Merck and Co., Inc. (1968 ed.) p 53.
RECEIVEDfor review February 17, 1976. Accepted April 7, 1976. This work solely supported by the U.S. Public Health
Comparisons between the Luminol Light Standards and a New Method for Absolute Calibrations of Light Detectors Paul R. Michael and Larry R. FaulkneP Department of Chemistry, University of Illinois, Urbana, Ill. 6 180 1
An actinometric technique for calibrating detectors used in the measurement of absolute light levels has been developed. It features a flexible light guide, which transmits monochromatic light from a mercury-xenon lamp to a sample cell contained in an integrating sphere. Measuring the quantum intensity from the guide, both actlnometrlcally and with a photomultiplier situated at the sphere's viewing port, allows one to calculate the absolute photometric calibration factor. The procedure also includes a relatively simple means for checking the wavelength dependence of the callbration factor. The results of the calibration were found to be consistent with the published liquid light standards based on the luminol chemiluminescent reactions in water and in DMSO. 1188
ANALYTICAL CHEMISTRY, VOL. 48, NO. 8, JULY 1976
The study of luminescent materials frequently is concerned with the quantum efficiency of a light emitting process. Determining it usually requires measurements of the total photon yield of a reaction over the entire wavelength range of its emission spectrum, which can span 100 nm or more (1,2).A sensitive detector such as a photomultiplier tube is usually required to detect the low light intensities generated in most luminescence experiments, and it must be calibrated in absolute units (e.g., incident photons per microcoulomb of integrated output current), so that the number of photons generated can be extracted from easily measured parameters. The calibration is complicated by the requirement that it be known for an extensive wavelength range in order to accom-
The use of sodium iodide as an external heavy atom perturber in the room temperature phosphorescence method has been investigated lor a wide varlety of compounds of biological and pharmaceutical Interest. In most cases, sodium iodide has been found to be very efficient in increasing the phosphorescence quantum yield and thus improved significantly the limits of detection of this rapid and simple method of molecular analysis.
Phosphorimetry has been developed into a sensitive spectrochemical method of analysis. Various recent developments and applications, especially in clinical chemistry, have been reviewed ( I , 2). In most phosphorimetric studies, the analysis has to be performed using either low temperature rigid matrices, such as organic glasses a t liquid nitrogen temperature or carefully-degassed solutions in order to minimize collisional triplet-quenching. The major analytical disadvantages of phosphorimetry are related to the need of cryogenic equipment or time-consuming degassing procedures, making this technique less-widely used than fluorimetry. Very recently, i t has been reported that intense phosphorescence was observed at room temperature of various salts of polynuclear organic compounds when adsorbed on filter paper, silica gel, alumina, and polycellulose (3-5). Room Temperature Phosphorimetry (RTP) has been thus suggested as a quantitative tative analytical method (6). Also, the external heavy atom effect on phosphorescence measured at room temperature has been reported with 2halonaphthalene sulfonate and some of its derivatives (7). Since the first report of solvent heavy-atom effect was given in 1952 by Kasha ( 8 ) ,who observed an increase in the oscilSo transition of l-chloronaphlator strength of the T I thalene by mixing it with ethyl iodide, it has been confirmed that the presence of heavy atoms, either as substituents (internal heavy-atom effect) or in the environment (external heavy-atom effect) can enhance significantly the process of intersystem crossing (9) and, therefore, the phosphorescence emission. In order to investigate the possibilities and improvements of this rapid and simple method of analysis, this work reports the external heavy-atom effect on phosphorescence a t room temperature. The samples selected for our investigation consist of a wide variety of compounds of biological and pharmaceutical interest.
-
EXPERIMENTAL Apparatus. The experimental set-up used in this study was previously described in detail (6).Phosphorescence measurements were performed with an Aminco-Bowman spectrophotofluorimeter with an Aminco-Keirs phosphoroscope attachment (American Instrument Co., Silver Spring, Md.). A 150-W xenon arc lamp was used as the excitation light source and the signal detected with a 1P21 photomultiplier tube with S-4 spectral response. Large monochromators slit widths (5 mm) were used for photometric detection limits, whereas higher spectral resolution was used for recording the spectra (1-mm monochromator slit width corresponding to 5-nm spectral resolution). The spectra were recorded on an x-y recorder (MFE Corp., Salem, N.H.).
Reagents. Eaton Dikeman 613 filter paper was used as support for all analytical investigations. Adenine, barbituric acid, guanine, 61186
ANALYTICAL CHEMISTRY, VOL. 48, NO. 8, JULY 1976
chloropurine, 6-methylmercaptopurine,sulfanilamide (Nutritional Biochemicals Corp., Cleveland, Ohio); L-(-)-tryptophan (Eastman Organic Chemicals, Rochester,N.Y.);p-aminobenzoicacid, salicyclic acid (Fisher Scientific Co., Fair Lawn, N.J.); cocaine hydrochloride (Applied Science Labs, Inc., State College, Pa.) were all used as received. Sodium hydroxide (Matheson Coleman and Bell, Norwood, Ohio) and sodium iodide (J. T. Baker Chemical Co., Phillipsburg, N.J.) were used to prepare the solvent. Procedure. Sample solutions were spotted on 0.25411. diameter filter paper circles and dried for about 30 min by infrared lamp heating. This technique has been shown to be more efficient than other methods using desiccators, blowers, or ovens. After the drying procedure, the samples were directly mounted on a simply designed holder (6). Since the phosphorescence emission is very sensitive to humidity, the sample compartment was continuously flushed with warm dried air. During all measurements, the temperature in the sample compartment was about 60 "C. RESULTS AND DISCUSSIONS Spectral Studies. The room temperature phosphorescence spectra of tryptophan adsorbed on filter paper from a 1 M NaOH aqueous solution (concentration = 6.6 X M) and a 1 M NaOH-1 M NaI solution (concentration = 7.3 x M) are shown in Figure 1. The presence of NaI greatly increases the observed phosphorescence signal; these two spectra in Figure 1 were recorded with different sensitivity scales. Obviously the heavy-atom enhancement technique affords considerable advantage in quantitative analysis since the limits of detection can be decreased, in some cases, by several orders of magnitude. Previously heavy metal ions, such as Hg2+ and Ag+, have been found to affect the phosphorescence of nucleosides and tryptophan a t 77 K ( 1 0 , l l ) .In the case of tryptophan, Hg2+ shortened the phosphorescence lifetime but also decreased the emission intensity, suggesting a quenching mechanism which might occur at the singlet levels (12).Ag+ metal ion has been found to increase substantially the phosphorescence intensity but, on the other hand, completely altered the vibrational structure. In the case of NaI used in R T P method, the emission intensity has been increased and the basic vibronic features were not significantly altered by the heavyatom perturbation; this indicated that the presence of NaI did not significantly change the nature of the emitting species and the radiative process. However, because of the low spectral resolution used in the present work, further work is needed prior to generalizations concerning the heavy-atom effect observed and the specific mechanism by which it occurs. In all measurements, the signals of the reference blanks showed an intensity increase of almost three times with NaI indicating that the paper emission is also affected by heavy-atom perturbation. Drying Conditions. The rigidity of the emitting species adsorbed on the paper support, and therefore the dryness of the sample is the main parameter affecting the phosphorescence emission intensity. The presence of moisture in the air can greatly reduce the phosphoresence intensity. After the paper sample has been exposed to the atmosphere and the moisture contained therein by removing the warm dry air supply, the phosphorescence signal decreased drastically. On the other hand, drying time has a reverse effect by increasing the phosphorescence intensity (Figure 2). Since the solutions contained a large excess of NaOH, in the process of drying the
'
t
n3
r
300
400
500
600
10
20
h(nrn)
30
40
50
TIME (min)
Figure 1. Tryptophan phosphorescence and the effect of Nal
Figure 2. Effect of drying time on room temperature phosphorescence intensity (2 X mol I.-' tryptophan in 1 M Na0H:l M Nal solution)
(1) Tryptophan in I M NaOH (c = 6.6 X mol I.-'). (2) Blank phosphorescence (paper with 1 M NaOH). (3)Tryptophan in 1 M Na0H:l M Nal ( c = 7 . 3 X mol I.-'), scale X 10. (4) Blank phosphorescence (paperwith 1 M Na0H:l M Nal), scale X 10. (5)Tryptophan phosphorescence at 77 K (from Ref. 74)
Curve 1. Phosphorescence intensity vs. drying time. Curve 2. Phosphorescence intensity vs. time after drying air has been removed.
(arbitrary scale)
sample on the paper support, the sample might become incorporated into a NaOH-Na2C03 matrix because of the presence of C02 in the drying air stream. The effect of Na2C03 as well as of various other experimental parameters on the phosphorescence intensity is of great interest and is now under investigation. The noteworthy feature is that the presence of oxygen (a known efficient triplet quencher in liquid solution a t room temperature) has no effect on the phosphorescence signal of dry samples observed by RTP. Even in polymer samples where large polyatomic molecules are immobilized in plastics, smaller molecules such as 0 2 can diffuse at sufficiently high rates to quench efficiently the phosphorescent triplets (13). All the measurements were performed after about 20 min, at which time the phosphorescence emission signal did not show any further increase in intensity. Analytical Curves. The analytical curve of tryptophan adsorbed on filter paper from a 1 M NaOH-1 M NaI (1:l) aqueous solution is reported in Figure 3. The slope of the log-lcg analytical curve is close to unity, and the range of
16
10-
rd 4
$63
CONCENTRATION(molar)
Figure 3. Room temperature phosphorescence analytical curve for tryptophan in 1 M Na0H:l M Nal solution adsorbed on filter paper at 60 O C
Table I. Phosphorimetric Limits of Detection (L.O.D.) for Various Compounds of Biological Importance AbsoluteC L.O.D.
Limits of detection"
Compound Adenine Guanine 6-Chloropurine 6-Methylmercaptopurine I,-(-)-Tryptophan Sulfanilamide Cocaine hydrochloride Para-aminobenzoic acid Salicylic acid Barbituric acid
Excitation wavelength, nm
Emission wavelength, nm
in NaOH, mol 1.-l
290 280 290 290
470 450 460 458
3.5 x 10-4 4.0 x 10-5 1.5 X lo-?
290 280 285
448 427 460
280
430
in NaOH:NaI, mol 1.-' 6.0 X 3.0 x 10-5 4.0 x 10-5 2.0 x 10-7
IY'*l
6.0 X 5.0 X
1.5 x 10-7 3.0 X loe6
40.0 1.7
4.5 x 10-5
3.0 x 10-5
1.5
7.0 x 10-7
9.0
10-5
x
10-8
Ip
1.6 12.0 1.0 0.8
12.0
ICng 2cng 6.8 4.1 264. 23. 31.
0.12
31. 0.17
6.1 0.15 4.3 2.6 76. 51. 0.48 0.062
1.0 x 10-6 4.0 2.8 0.69 5.0 x 10-5 1.4 45. 32. Absolute limits of optical detection, See text for definitions. a Defined as that concentration resulting in a signal-to-noiseratio of 2. calculated for 5 - ~ sample 1 solutions, ICin NaOH solution, 2c in Na0H:NaI solution.
320 290
470 455
4.0 7.0
X
x 10-5
ANALYTICAL CHEMISTRY, VOL. 48, NO. 8, JULY 1976
1187
linearity is fairly large (-lo3). Compared with data of tryptophan in a solvent without NaI, the analytical linearity range has been increased by a t least an order of magnitude because of the decrease of the detection limit at the low concentrations range. Because it was observed that the compounds could be gradually decomposed by the strong basic solvent, all the measurements were performed using freshly-prepared samples. Limits of Phosphorimetric Detection. Table I reports the limits of phosphorimetric detection of a wide variety of compounds of biological importance. The sodium iodide heavy-atom enhancement factor IpN"'/Ip is defined as the ratio of the molar phosphorescence signal in 1 M NaOH-1 M NaI (1:l) solution to that in 1M NaOH aqueous solution, respectively, and was deduced from the following equation:
where ip*, ,'?i c p ' are, respectively, the phosphorescence signals (in A) of the sample, S, the reference blank, R, and the concentration (mol l.-I) of the substance, S, under investigation in a solution of NaOH-NaI; and the other parameters correspond to those items for only NaOH solution (no NaI). Except for 6-methylmercaptopurine, the heavy-atom enhancement factor was found for most compounds to be greater than unity. It ranged from 1.0 for 6-chloropurine to 12 for guanine, and 40 for tryptophan. The data show that the heavy-atom effect on room temperature phosphorescence varies appreciably with the molecular structure of the compounds and the nature of the excited states involved. Advantages of R T P f o r Analysis. An obvious advantage of R T P technique using paper for analytical purposes, besides its simplicity, is the small amount of sample which is required for every measurement; i.e., only 5 ~1 is usually needed to prepare a sample, whereas low temperature phosphorimetry usually requires 100 ~1 to 1ml and conventional fluorimetry requires as much as 4 ml. Therefore, the absolute sensitivity (in ng) of R T P is in some cases much higher than the con-
ventional techniques; for tryptophan, an absolute limit of detection of 0.15 ng can be achieved. The significant enhancement of tryptophan phosphorescence is of special interest in protein analysis since most proteins have been found to emit via their tryptophan residues. Para-aminobenzoic (PABA) acid which has a very low limit of detection (9 X mol l.-l) is also of medical interest because it has been used to raise blood salicylate level, or in the laboratory as a sulfonamide antagonist, or more commonly as a sunscreen agent (14). The absolute limit of phosphorimetric detection of PABA is 0.06 ng. This study has shown that the external heavy-atom effect on room temperature phosphorescence is applicable to a wide variety of compounds of biological importance. In some cases, the sensitivity of phosphorimetric detection is increased by several orders of magnitude. It is therefore suggested that the use of the external heavy-atom effect be applied to improve the sensitivity of room temperature phosphorimetric method of analysis. This technique, which is a rapid, simple method of analysis, would be quite suitable for clinical analysis. LITERATURE CITED (1) C. M. O'Donnell and J. D. Winefordner, Ciin. Chem., ( Winston-Salem,N.C.), 21, 285 (1975). (2) J. J. Aaron and J. D. Winefordner, Talanfa, 22, 707 (1975). (3) E. M. Schulman and C. Walling, Science, 178, 53 (1972). (4) E. M. Schulman and C. Walling, J. fhys. Chem., 77, 902 (1973). (5) P. G. Seybold, R. K.Sorrel1 and Schuffert, 165th National Meeting, American Chimical Society, Dallas, Texas, April 13, 1973. (6) S. L. Wellons, R. A. Paynter, and J. D. Winefordner, Spectrochim. Acta, fartA, 30, 2133 (1974). (7) P. G. Seybold and W. White, Anal. Chem., 47, 1199 (1975). (8) M. Kasha, J. Chem. fhys., 20, 71 (1952). (9) T. Medinger and F. Wllkinson, Trans. Farad. Soc., 61, 620 (1965). (10) R. F. Chen, Anal. Biochem. Biophys., 144, 552 (1971). (11) G. D. Boutilier, C. M. O'Donnell, and R. 0. Rahn, Anal. Chem., 46, 1508 (1974). (12) R . F. Chen, Amhco Fiuorescence News/., 9, No. 2 and 3, 9 (1975). (13) B. A. Baldwin and H. W. Otfen, J. Chem. fhys., 49, 2933 (1968). (14) The Merck Index, Merck and Co., Inc. (1968 ed.) p 53.
RECEIVEDfor review February 17, 1976. Accepted April 7, 1976. This work solely supported by the U.S. Public Health
Comparisons between the Luminol Light Standards and a New Method for Absolute Calibrations of Light Detectors Paul R. Michael and Larry R. FaulkneP Department of Chemistry, University of Illinois, Urbana, Ill. 6 180 1
An actinometric technique for calibrating detectors used in the measurement of absolute light levels has been developed. It features a flexible light guide, which transmits monochromatic light from a mercury-xenon lamp to a sample cell contained in an integrating sphere. Measuring the quantum intensity from the guide, both actlnometrlcally and with a photomultiplier situated at the sphere's viewing port, allows one to calculate the absolute photometric calibration factor. The procedure also includes a relatively simple means for checking the wavelength dependence of the callbration factor. The results of the calibration were found to be consistent with the published liquid light standards based on the luminol chemiluminescent reactions in water and in DMSO. 1188
ANALYTICAL CHEMISTRY, VOL. 48, NO. 8, JULY 1976
The study of luminescent materials frequently is concerned with the quantum efficiency of a light emitting process. Determining it usually requires measurements of the total photon yield of a reaction over the entire wavelength range of its emission spectrum, which can span 100 nm or more (1,2).A sensitive detector such as a photomultiplier tube is usually required to detect the low light intensities generated in most luminescence experiments, and it must be calibrated in absolute units (e.g., incident photons per microcoulomb of integrated output current), so that the number of photons generated can be extracted from easily measured parameters. The calibration is complicated by the requirement that it be known for an extensive wavelength range in order to accom-
